Devices and techniques associated with diagnostics, therapies, and other applications, including skin-associated applications

ABSTRACT

The present invention generally relates to devices and techniques associated with diagnostics, therapies, and other applications, including skin-associated applications, for example, devices for delivering and/or withdrawing fluid from subjects, e.g., through the skin. In some embodiments, the device includes a system for accessing an extractable medium from and/or through the skin of the subject at an access site, and a pressure regulator supported by a support structure, able to create a pressure differential across the skin at at least a portion of the access site. The device may also include, in some cases, a sensor supported by the support structure for determining at least one condition of the extractable medium from the subject, and optionally a signal generator supported by the support structure for generating a signal relating to the condition of the medium determined by the sensor.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/163,710, filed Mar. 26, 2009, entitled “Systemsand Methods for Creating and Using Suction Blisters or Other PooledRegions of Fluid within the Skin,” by Levinson, et al.; U.S. ProvisionalPatent Application Ser. No. 61/156,632, filed Mar. 2, 2009, entitled“Oxygen Sensor,” by Levinson, et al.; U.S. Provisional PatentApplication Ser. No. 61/269,436, filed Jun. 24, 2009, entitled “Devicesand Techniques associated with Diagnostics, Therapies, and OtherApplications, Including Skin-Associated Applications,” by Levinson, etal.; U.S. Provisional Patent Application Ser. No. 61/257,731, filed Nov.3, 2009, entitled “Devices and Techniques associated with Diagnostics,Therapies, and Other Applications, Including Skin-AssociatedApplications,” by Bernstein, et al.; and U.S. Provisional PatentApplication Ser. No. 61/294,543, filed Jan. 13, 2010, entitled “BloodSampling Device and Method,” by Levinson, et al. Each of the above isincorporated herein by reference.

FIELD OF INVENTION

The present invention generally relates to interstitial and other bodilyfluids for use in analyzing a condition of a subject or treating asubject. The present invention also relates to systems and methods fordelivering and/or withdrawing fluid from subjects, e.g., through theskin.

BACKGROUND

A variety of techniques and methods exist for sensing and responding toconditions to which a subject is exposed, including sensing ofphysiological conditions of a mammal and/or a surrounding environment.Other techniques exist for delivering and/or withdrawing a fluid from amammal, such as blood. While many such techniques are suitable forvarious purposes, techniques that have one or more features such asadded simplicity and flexibility of use would be advantageous.

SUMMARY OF THE INVENTION

The present invention, in some aspects, generally relates tointerstitial and other bodily fluids (e.g., blood) for use in analyzinga condition of a subject or treating a subject. In some cases, thepresent invention also relates to systems and methods for deliveringand/or withdrawing fluid from subjects, e.g., through the skin. Thesubject matter of the present invention involves, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of one or more systems and/orarticles.

The present invention is directed to a device for analysis of anextractable medium from a subject in one aspect. In one set ofembodiments, the device includes a support structure, means associatedwith the support structure for accessing the extractable medium fromand/or through the skin of the subject at an access site, and a pressureregulator supported by the support structure, able to create a pressuredifferential across the skin at at least a portion of the access site.In some cases, the device may also include a sensor supported by thesupport structure, for determining at least one condition of theextractable medium from the subject, and optionally a signal generatorsupported by the support structure, for generating a signal relating tothe condition of the medium determined by the sensor. In someembodiments, the support structure is constructed and arranged to bepositioned proximate the skin of the subject.

In another set of embodiments, the device includes a support structureconstructed and arranged to be positioned proximate the skin of thesubject, a fluid transporter for accessing the extractable medium fromand/or through the skin of the subject at an access site, a pressureregulator supported by the support structure, able to create a pressuredifferential across the skin at at least a portion of the access site, asensor supported by the support structure for determining at least onecondition of the extractable medium from the subject, and a signalgenerator supported by the support structure for generating a signalrelating to the condition of the medium determined by the sensor.

The device, according to yet another set of embodiments, includes meansfor accessing the extractable medium from and/or through the skin of thesubject at an access site, a pressure regulator able to create apressure differential across the skin at at least a portion of theaccess site, a sensor for determining at least one condition of theextractable medium from the subject, and a signal generator forgenerating a signal relating to the condition of the medium determinedby the sensor.

In another set of embodiments, the device includes means for accessingthe extractable medium from skin and/or from beneath the skin of thesubject at an access site, a pressure differential chamber able tocreate a pressure differential across the skin in at least a portion ofthe access site in the absence of a piston pump associated with thechamber, a sensor for determining at least one condition of theextractable medium from the subject, and a signal generator forgenerating a signal relating to the condition of the medium determinedby the sensor.

The invention, in another aspect, is generally directed to a device.According to one set of embodiments, the device can be applied or isapplicable to the skin for determining an analyte in a subject. Thedevice, in one embodiment, includes a first portion able to create apooled region of fluid within the skin of a subject, and a secondportion able to determine fluid of the pooled region of fluid. Inanother set of embodiments, the device includes a first portion able tocreate a pooled region of fluid within the skin of a subject, and amicrofluidic channel in fluidic communication with the first portion.

The device, in yet another set of embodiments, is applicable to a siteproximate the skin of a subject. In some cases, the device is able todetermine a physical condition of a subject and produces a signalrelated to the condition. In one embodiment, the signal is not readilyunderstandable by the subject.

The device, in yet another set of embodiments, is a device applicable toa site proximate the skin of a subject. In some embodiments, the deviceis able to determine a physical condition of the subject. In oneembodiment, the device comprises an agent that reacts or interacts withan analyte to be detected and generates a signal that can be detectedvisually, by feel, by smell, or by taste. In some cases, the agent thatreacts with or interacts with an analyte and the signaling agent can bethe same or different. In certain cases, the signal can vary as afunction of an attribute of the analyte, producing a signal that can beanalyzed qualitatively. In some instances, the device acquires fluidfrom the subject and transports the fluid from a site of acquisition toa site of analysis for reaction.

In some cases, the device includes a first component and a secondcomponent connected to the first component for application to the siteand separable from the first component after application to the site. Insome instances, the first component can be activated to acquire a bodilyfluid of the subject, and the second component comprises an agent thatreacts or interacts with an analyte to be detected and generates asignal that can be detected visually, by feel, by smell, or by taste.

The device, in accordance with another set of embodiments, includes anagent that reacts or interacts with an analyte in saliva of the subjectto generate a signal.

In still another set of embodiments, the device comprises an agent thatreacts or interacts with an analyte to be detected to generate a signalthat can be detected visually, by feel, by smell, or by taste. In somecases, the device comprises a membrane comprising the agent thatparticipates in generation of the signal. The membrane, in someembodiments, may be selective to one or more of passage of an analyteand/or an analyte indicator, and reaction with an analyte and/or ananalyte indicator.

According to still another set of embodiments, the device is a devicefor withdrawing blood from the skin and/or from beneath the skin of asubject. In some embodiments, the device comprises a fluid transporter,a vacuum chamber having an internal pressure less than atmosphericpressure before blood is withdrawn into the device, and a storagechamber, separate from the vacuum chamber, for receiving blood withdrawnfrom the subject through the fluid transporter when a negative pressureis applied to the skin of the subject. The device, in certainembodiments, includes at least 6 microneedles, and a storage chamber forreceiving blood withdrawn from the subject, the storage chamber havingan internal pressure less than atmospheric pressure prior to receivingblood.

The device, in some embodiments, includes a fluid transporter, a firststorage chamber for receiving blood withdrawn from the subject throughthe fluid transporter, the storage chamber having an internal pressureless than atmospheric pressure prior to receiving blood, and a reactionentity contained within the first storage chamber able to react with ananalyte contained within the blood. In some instances, a product of thereaction entity with the analyte is determinable. According to stillother embodiments, the device includes a fluid transporter, a storagechamber for receiving blood withdrawn from the subject through the fluidtransporter, and a potassium sensor able to determine potassium ionswithin blood contained within the device. In some cases, the storagechamber has an internal pressure less than atmospheric pressure prior toreceiving blood.

In yet another set of embodiments, the device includes a fluidtransporter, a storage chamber for receiving blood withdrawn from thesubject through the fluid transporter, and a flow controller able tocontrol blood flow into the storage chamber. In some cases, the storagechamber has an internal pressure less than atmospheric pressure prior toreceiving blood.

In another aspect, the present invention is directed to a method. Themethod, in one set of embodiments, includes acts of causing formation ofa pooled region of fluid between the dermis and epidermis of the skin ofa subject, and delivering an agent into the pooled region of fluid forindication of a past, present and/or future condition of the subject.The method, according to another set of embodiments, includes acts ofcausing formation of a pooled region of fluid between the dermis andepidermis of the skin of a subject, removing at least a portion of thefluid from the pooled region, and analyzing at least a portion of theremoved fluid thereby determining a past, present and/or futurecondition of the subject by exposing the fluid to an agent.

The method, according to another set of embodiments, includes acts ofproviding a device at a site proximate the skin of a subject, acquiringa representation of the signal, and transporting the representation ofthe signal to an entity that analyzes the representation. In some cases,the device can determine a physical condition of a subject and producesa signal related to the condition. In yet another set of embodiments,the method includes an act of applying, proximate the skin of a subjectin need of emergency care, a device comprising a reactive agent thatreacts or interacts with an analyte to generate a signal indicative of acondition of the subject. The method, in still another set ofembodiments, includes an act of applying, to a solution containing aplurality of first particles of a first color and a plurality of secondparticles of a second color, an electric field and/or a magnetic fieldsufficient to cause a change in color of the solution. According to yetanother set of embodiments, the method includes an act of determining acharacteristic of a sample by visualizing a color change effected by achange in population of particles of different color upon preferentialbinding of particles of one color to a component of the sample via abinding partner.

The method, in one set of embodiments, includes acts of exposing, to asample, a population of particles including at least a firstsub-population having a first determinable characteristic and a secondsub-population having a second determinable characteristic, allowing atleast some particles of the first sub-population of particles to bind acomponent of the sample via a binding partner, separating the particlesbound to the component from those not bound, and determining acharacteristic of the sample by determining the separation. In someembodiments, the second sub-population of particles remains essentiallyunbound to the component, such that the ratio of the number of particlesfrom the first sub-population bound to the component, to the number ofparticles from the second sub-population bound to the component, is atleast 5:1.

In another set of embodiments, the method includes an act of providing aplurality of analysis sites proximate the skin of a subject, includingat least a first analysis site that generates a first signal related toa first physical condition of the subject and a second analysis sitethat generates a second signal related to a second physical condition ofthe subject. The method, in accordance with still another set ofembodiments, includes an act of providing a reaction site proximate theskin of a subject comprising a reactive agent that reacts or interactswith an analyte to generate an signal in the form of an icon.

In yet another set of embodiments, the method is a method for analyzingan extractable medium from a subject. In some embodiments, the methodincludes acts of positioning an analysis device comprising a pathwayadjacent the skin of the subject and, while the device is adjacent theskin, activating an access component of the device to connect theextractable medium with the pathway of the device, activating a pressurecontroller of the device to urge the extractable medium into the devicevia the pathway, exposing the medium to a sensor of the device anddetermining at least one condition of the extractable medium from thesubject, and generating a signal relating to the condition of the mediumdetermined by the sensor.

In another aspect, the present invention is directed to a method ofmaking one or more of the embodiments described herein. In anotheraspect, the present invention is directed to a method of using one ormore of the embodiments described herein.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1E schematically illustrate various systems and methods forcreating pooled regions of fluid in the skin, according to certainembodiments of the invention;

FIGS. 2A-2C illustrate various devices useful for creating pooledregions of fluid in the skin, in some embodiments;

FIGS. 3A-3C illustrate an example of one embodiment of the inventionuseful for puncturing and creating a pooled region of the skin;

FIGS. 4A-4B illustrate additional examples of various devices of theinvention;

FIG. 5 illustrates a check valve according to one embodiment of theinvention;

FIG. 6 illustrates certain non-circular interfaces, in accordance withanother embodiment of the invention;

FIG. 7 illustrates another embodiment of the invention useful forcreating a pooled region of the skin;

FIGS. 8A-8B illustrates top and side views, respectively, of apre-loaded vacuum chamber, in another embodiment of the invention;

FIGS. 8C-8E illustrate a device containing a compressed foam, in yetanother embodiment of the invention;

FIG. 9A-9B illustrate devices according to certain embodiments of theinvention;

FIGS. 10A-10B illustrate devices according to various embodiments of theinvention;

FIG. 11A illustrates a device according to still another embodiment ofthe invention;

FIG. 11B illustrates a kit containing more than one device, in yetanother embodiment of the invention; and

FIG. 11C illustrates a device according to still another embodiment ofthe invention.

DETAILED DESCRIPTION

The present invention generally relates to devices and techniquesassociated with diagnostics, therapies, and other applications,including skin-associated applications, for example, devices fordelivering and/or withdrawing fluid from subjects, e.g., through theskin. In some embodiments, the device includes a system for accessing anextractable medium from and/or through the skin of the subject at anaccess site, and a pressure regulator supported by a support structure,able to create a pressure differential across the skin at at least aportion of the access site. The device may also include, in some cases,a sensor supported by the support structure for determining at least onecondition of the extractable medium from the subject, and optionally asignal generator supported by the support structure for generating asignal relating to the condition of the medium determined by the sensor.

Non-limiting examples of various devices of the invention are shown inFIG. 9. In FIG. 9A, device 290 is used for withdrawing a fluid from asubject when the device is placed on the skin of a subject. Device 290includes sensor 295 and fluid transporter 292, e.g., a needle, amicroneedle, etc., as discussed herein. In fluidic communication withfluid transporter 292 via fluidic channel 299 is sensing chamber 297. Inone embodiment, sensing chamber 297 may contain agents such asparticles, enzymes, dyes, etc., for analyzing bodily fluids, such asinterstitial fluid or blood. In some cases, fluid may be withdrawn usingfluid transporter 292 by a vacuum, for example, a self-contained vacuumcontained within device 290. Optionally, device 290 also contains adisplay 294 and associated electronics 293, batteries or other powersupplies, etc., which may be used to display sensor readings obtainedvia sensor 295. In addition, device 290 may also optionally containmemory 298, transmitters for transmitting a signal indicative of sensor295 to a receiver, etc.

As used herein, “fluid transporter” is any component or combination ofcomponents that facilitates movement of a fluid from one portion of thedevice to another. For example, at or near the skin, a fluid transportercan be a hollow needle when a hollow needle is used or, if a solidneedle is used, then if fluid migrates along the needle due to surfaceforces (e.g., capillary action), then the solid needle can be a fluidtransporter. If fluid (e.g. blood or interstitial fluid) partially orfully fills an enclosure surrounding a needle after puncture of skin(whether the needle is or is not withdrawn from the skin afterpuncture), then the enclosure can define a fluid transporter. Othercomponents including partially or fully enclosed channels, microfluidicchannels, tubes, wicking members, vacuum containers, etc. can be fluidtransporters

In the example shown in FIG. 9A, device 290 may contain a vacuum source(not shown) that is self-contained within device 290, although in otherembodiments, the vacuum source may be external to device 290. (In stillother instances, other systems may be used to deliver and/or withdrawfluid from the skin, as is discussed herein.) In one embodiment, afterbeing placed on the skin of a subject, the skin may be drawn upward intoa recess containing fluid transporter 292, for example, upon exposure tothe vacuum source. Access to the vacuum source may be controlled by anysuitable method, e.g., by piercing a seal or a septum; by opening avalve or moving a gate, etc. For instance, upon activation of device290, e.g., by the subject, remotely, automatically, etc., the vacuumsource may be put into fluidic communication with the recess such thatskin is drawn into the recess containing fluid transporter 292 due tothe vacuum. Skin drawn into the recess may come into contact with fluidtransporter 292 (e.g., solid or hollow needles), which may, in somecases, pierce the skin and allow a fluid to be delivered and/orwithdrawn from the skin. In another embodiment, fluid transporter 292may be actuated and moved downward to come into contact with the skin,and optionally retracted after use.

Another non-limiting example of a device is shown in FIG. 9B. Thisfigure illustrates a device useful for delivering a fluid to thesubject. Device 290 in this figure includes fluid transporter 292, e.g.,a needle, a microneedle, etc., as discussed herein. In fluidiccommunication with fluid transporter 292 via fluidic channel 299 ischamber 297, which may contain a drug or other agent to be delivered tothe subject. In some cases, fluid may be delivered with a pressurecontroller, and/or withdrawn using fluid transporter 292 by a vacuum,for example, a self-contained vacuum contained within device 290. Forinstance, upon creating a vacuum, skin may be drawn up towards fluidtransporter 292, and fluid transporter 292 may pierce the skin. Fluidfrom chamber 297 can then be delivered into the skin through fluidchannel 299 and fluid transporter 292. Optionally, device 290 alsocontains a display 294 and associated electronics 293, batteries orother power supplies, etc., which may be used control delivery of fluidto the skin. In addition, device 290 may also optionally contain memory298, transmitters for transmitting a signal indicative of device 290 orfluid delivery to a receiver, etc.

Yet another non-limiting example of a device of the invention is shownin FIG. 10. FIG. 10A illustrates a view of the device (with the coverremoved), while FIG. 10B schematically illustrates the device incross-section. In FIG. 10B, device 250 includes a needle 252 containedwithin a recess 255. Needle 252 may be solid or hollow, depending on theembodiment. Device 250 also includes a self-contained vacuum chamber260, which wraps around the central portion of the device where needle252 and recess 255 are located. A channel 262 connects vacuum chamber260 with recess 255, separated by foil or a membrane 267. Also shown indevice 250 is button 258. When pushed, button 258 breaks foil 267,thereby connecting vacuum chamber 250 with recess 255, creating a vacuumin recess 255. The vacuum may be used, for example, to draw skin intorecess 255, preferably such that it contacts needle 252 and pierces thesurface, thereby gaining access to an internal fluid. The fluid may becontrolled, for example, by controlling the size of needle 252, andthereby the depth of penetration. For example, the penetration may belimited to the epidermis, e.g., to collect interstitial fluid, or to thedermis, e.g., to collect blood. In some cases, the vacuum may also beused to at least partially secure device 250 on the surface of the skin,and/or to assist in the withdrawal of fluid from the skin. For instance,fluid may flow into channel 262 under action of the vacuum, andoptionally to sensor 261, e.g., for detection of an analyte containedwithin the fluid. For instance, sensor 261 may produce a color change ifan analyte is present, or otherwise produce a detectable signal.

Other components may be added to the example of the device illustratedin FIG. 10, in some embodiments of the invention. For example, device250 may contain a cover, displays, ports, transmitters, sensors,microfluidic channels, chambers, fluid channels, and/or variouselectronics, e.g., to control or monitor fluid transport into or out ofdevice 250, to determine an analyte present within a fluid deliveredand/or withdrawn from the skin, to determine the status of the device,to report or transmit information regarding the device and/or analytes,or the like, as is discussed in more detail herein. As another example,device 250 may contain an adhesive, e.g., on surface 254, for adhesionof the device to the skin.

Yet another non-limiting example is illustrated with reference to FIG.11A. In this example, device 500 includes a support structure 501, andan associated fluid transporter system 503. Fluid transporter system 503includes a plurality of needles or microneedles 505, although otherfluid transporters as discussed herein may also be used. Also shown inFIG. 11A is sensor 510, connected via channels 511 to recess 508containing needles or microneedles 505. Chamber 513 may be aself-contained vacuum chamber, and chamber 513 may be in fluidiccommunication with recess 508 via channel 511, for example, ascontrolled by a controller or an actuator (not shown). In this figure,device 500 also contains display 525, which is connected to sensor 510via electrical connection 522. As an example of use of device 500, whenfluid is drawn from the skin (e.g., blood, interstitial fluid, etc.),the fluid may flow through channel 511 to be determined by sensor 510,e.g., due to action of the vacuum from vacuum chamber 513. In somecases, the vacuum is used, for example, to draw skin into recess 508,e.g., such that it contacts needles or microneedles 505 and pierces thesurface of the skin to gain access to a fluid internal of the subject,such as blood or interstitial fluid, etc. The fluid may be controlled,for example, by controlling the size of needle 505, and thereby thedepth of penetration. For instance, the penetration may be limited tothe epidermis, e.g., to collect interstitial fluid, or to the dermis,e.g., to collect blood. Upon determination of the fluid and/or ananalyte present or suspected to be present within the fluid, amicroprocessor or other controller may display a suitable signal ondisplay 525. As is discussed below, a display is shown in this figure byway of example only; in other embodiments, no display may be present, orother signals may be used, for example, lights, smell, sound, feel,taste, or the like. In some cases, more than one fluid transportersystem may be present within the device. For instance, the device may beable to be used repeatedly, and/or the device may be able to deliverand/or withdraw fluid at more than one location on a subject, e.g.,sequentially and/or simultaneously. In some cases, the device may beable to simultaneously deliver and withdraw fluid to and from a subject.A non-limiting example of a device having more than one fluidtransporter system is illustrated with reference to FIG. 11C. In thisexample, device 500 contains a plurality of structures such as thosedescribed herein for delivering to and/or withdrawing fluid from asubject. For example, device 500 in this example contains 3 such units,although any number of units are possible in other embodiments. In thisexample, device 500 contains three such fluid transporter systems 575.Each of these fluid transporter systems may independently have the sameor different structures, depending on the particular application, andthey may have structures such as those described herein.

As noted, the invention in one set of embodiments involves determinationof a condition of a subject. In such a case, bodily fluid and/ormaterial associated with the skin may be analyzed, for instance, as anindication of a past, present and/or future condition of the subject, orto determine conditions that are external to the subject. Determinationmay occur, for instance, visually, tactilely, by odor, viainstrumentation, etc. Other aspects of the invention are directed todevices able to create pooled regions within the skin, and areoptionally able to remove fluid from the pooled region and/or addmaterial to the pooled region. Still other aspects of the invention aregenerally directed to making or using such devices, methods of promotingthe making or use of such devices, and the like.

Agents such as particles can be used to analyze bodily fluids, such asinterstitial fluid or blood. Other suitable agents include enzymes,dyes, nucleic acids, antibodies, oligonucleotides, reaction entities, orthe like, e.g., as discussed herein. The interstitial fluids may beaccessed or collected using a suction blister device in certainembodiments, or using other techniques such as those described below.The agent may be any agent able to determine an analyte. For example,the agent may be an antibody which is labeled with a colorimetric, gold,or fluorescent label, which binds analyte, producing a color changewhich is proportional to the amount of analyte. In some cases, more thanone agent may be used. For example, a first agent may react with ananalyte and the second agent may be used to create a determinablesignal, for instance, visual, tactile, smell, taste, shape change, orthe like. Combinations of these can also be utilized in some cases. Forexample, an antibody to a carcinoembryonic antigen (“CEA”) and anantibody to a prostate specific antigen (“PSA”) may be used to monitorfor cancer of either origin; the antibodies may be used to producevarious colors upon detection of their corresponding analytes, forinstance, the colors may be yellow for CEA and blue for PSA, resultingin green if both are elevated.

In one set of embodiments, one or more of the agents may be particles,such as anisotropic particles or colloids. Accordingly, in thedescriptions that follow, it should be understood that “particles” aredescribed by way of example only, and in other embodiments, otheragents, such as antibodies, may also be used, in addition and/or insteadof particles.

In other embodiments, fluid may be delivered to the subject, and suchfluids may contain materials useful for delivery, e.g., forming at leasta portion of the fluid, dissolved within the fluid, carried by the fluid(e.g., suspended or dispersed), or the like. Examples of suitablematerials include, but are not limited to, particles such asmicroparticles or nanoparticles, a chemical, a drug or a therapeuticagent, a diagnostic agent, a carrier, or the like.

As used herein, the term “fluid” generally refers to a substance thattends to flow and to conform to the outline of its container. Typically,fluids are materials that are unable to withstand a static shear stress,and when a shear stress is applied, the fluid experiences a continuingand permanent distortion. The fluid may have any suitable viscosity thatpermits at least some flow of the fluid. Non-limiting examples of fluidsinclude liquids and gases, but may also include free-flowing solidparticles, viscoelastic fluids, and the like. For example, the fluid mayinclude a flowable matrix or a gel, e.g., formed from biodegradableand/or biocompatible material such as polylactic acid, polyglycolicacid, poly(lactic-co-glycolic acid), etc., or other similar materials.

One set of embodiments generally relates to interstitial and otherbodily fluids for use in analyzing a condition of a subject or treatinga subject. For instance, certain aspects of the invention are directedto causing the formation of a pooled region of fluid between the dermisand epidermis, such as in a suction blister. Fluid may be removed fromthe pooled region and analyzed in some fashion, or material may bedelivered to the pooled region of fluid, in either case for diagnosisand/or treatment of a condition of a subject. In one set of embodiments,various particles may be delivered to the fluid, whether pooled betweenthe dermis or epidermis, or in fluid removed from the subject, and theparticles can assist in diagnosis or treatment as described herein.Optionally, fluid within a pooled region may be drained, e.g.,externally, or the fluid may be resorbed, which may leave particles orother material embedded within the skin between the epidermis anddermis.

In some cases, as discussed herein, pooled regions of fluid may becreated in the skin for facilitating delivery and/or withdraw of fluidfrom the skin. For instance, fluid may be pooled within the skin that isdrawn from the surrounding dermal and/or epidermal layers within theskin. The fluid may include interstitial fluid, or even blood in somecases. In other cases, however, no pooling is necessary for the deliveryand/or withdraw of fluid from the skin. For example, fluid may bewithdrawn from the skin of a subject into a device, for example, usingvacuum or other techniques such as discussed below, and/or fluid may bedelivered into the skin of a subject without necessarily needing tocreate a suction blister or a pooled region of fluid in which to deliverfluid. Accordingly, it should be understood that in the descriptionsherein, references to a “suction blister” or “pooled region of fluid”are by way of example only, and in other embodiments, fluid may bedelivered to the skin without using a suction blister and/or withoutcreating a pooled region of fluid within the skin.

It should also be understood that, in some cases, fluid may be createdbeneath the skin, e.g., in the fatty or muscle layers below the skin.Accordingly, descriptions herein of delivering and/or withdrawing fluid“in the skin” should also be understood to include, in otherembodiments, the deliver and/or withdraw of fluid into layers directlybeneath the skin.

One embodiment of the invention is illustrated in FIG. 1. In FIG. 1A,the skin 10 of a subject is shown, having two layers: an upper epidermislayer 15, and a lower dermis layer 17. Of course, other structures aretypically present within the skin, for example, blood vessels, nerveendings, sweat glands, hair follicles, or the like, but these are notillustrated here for clarity. In FIG. 1B, epidermis 15 has beenpartially separated from dermis 17, creating a region 20 within the skinbetween the epidermis and the dermis. This region typically fills withfluid, such as interstitial fluid from the body, forming a pooled regionof fluid. As shown in the example of FIG. 1B, the separation of thedermis and epidermis is created using interface 25 that is able to applyvacuum to the surface of the skin, thereby creating a suction blisterwithin the skin formed by the pooled region of fluid. For example,vacuum may be created via conduit 27, which may be in fluidiccommunication with a vacuum source, such as a vacuum pump or an external(line) vacuum source. However, as discussed below, other methods may beused to create a pooled region of fluid within the skin besides, or inaddition to, the use of vacuum. In addition, as previously discussed,certain embodiments of the invention do not require the creation of apooled region of fluid within the skin.

FIG. 1C shows an embodiment in which fluid is removed from pooled region20. In this figure, needle 35, such as a hypodermic needle, may be usedto remove at least a portion of the fluid from pooled region 20. Thefluid may be stored, and/or analyzed to determine one or more analytes,e.g., a marker for a disease state, or the like. In some cases, thefluid may be actively removed, for example, upon the application ofvacuum to the surface of skin 10, or upon the application of hygroscopicmaterial to facilitate the removal of fluid from pooled region 20. Inother cases, the fluid may be extracted, for example, via vacuum appliedto the surface of skin 10 or to needle 35. In other cases, the fluid maybe allowed to leave pooled region 20 passively. For example, in oneembodiment, a cutter may be used to create a conduit by which fluid frompooled region 20 can escape. The cutter may be, for example, a needle orother piercing instrument, a cutting blade, or the like.

In FIG. 1D, material 29 is shown being delivered into the pooled regionof fluid 20. Material 29 may be, for example, particles, such asmicroparticles or nanoparticles, a chemical, a drug or a therapeuticagent, a diagnostic agent, a reaction entity, or the like. In somecases, material 29 may comprise a carrier, such as a fluid carrier, thatcomprises the particles, chemicals, drugs, therapeutic agents,diagnostic agents, reaction entities, etc. As shown in this figure,needle 32 is used to deliver material 29 into pooled region 20. However,in other embodiments, other systems, such as a jet injector, may be usedto deliver material 29 into the pooled region 20.

Once needle 32 has been removed from skin 10, material 29 may becomeembedded within pooled region 20, or may be dispersed within thesubject, depending on the nature of material 29. For example, if fluidwithin the pooled region of fluid 20 is drained in some fashion, e.g.,externally or is resorbed, etc., material 29 may remain embedded betweenepidermis 15 and dermis 17, as is shown in FIG. 1E. The material maybecome temporarily or permanently embedded between the dermis and theepidermis, or in some cases, the material may disperse within thesubject, for example, dissolving or biodegrading within the subject, orthe material may be one that can be transported within the subject, suchas within the bloodstream. For example, in some cases, material 29 maycontain a drug or other therapeutic agent that can be released frommaterial 29, e.g., upon degradation of material 29. In one embodiment,for instance, material 29 may comprise particles able to controllablyrelease a drug, a diagnostic agent, a therapeutic agent, etc.

Thus, certain aspects of the present invention are generally directed tothe creation of suction blisters or other pooled regions of fluid withinthe skin. In one set of embodiments, a pooled region of fluid can becreated between the dermis and epidermis of the skin. Suction blistersor other pooled regions may form in a manner such that the suctionblister or other pooled region is not significantly pigmented in somecases, since the basal layer of the epidermis contains melanocytes,which are responsible for producing pigments. Such regions can becreated by causing the dermis and the epidermis to at least partiallyseparate, and as will be discussed below, a number of techniques can beused to at least partially separate the dermis from the epidermis. Asmentioned, however, some embodiments of the invention do not necessarilyrequire the creation of a pooled region of fluid within the skin.

The subject is usually human, although non-human subjects may be used incertain instances, for instance, other mammals such as a dog, a cat, ahorse, a rabbit, a cow, a pig, a sheep, a goat, a rat (e.g., RattusNorvegicus), a mouse (e.g., Mus musculus), a guinea pig, a hamster, aprimate (e.g., a monkey, a chimpanzee, a baboon, an ape, a gorilla,etc.), or the like.

In one technique, a pool of interstitial fluid is formed between layersof skin of a subject and, after forming the pool, fluid is drawn fromthe pool by accessing the fluid through a layer of skin, for example,puncturing the outer layer of skin with a needle, e.g., with amicroneedle. Specifically, for example, a suction blister can be formedand then the suction blister can be punctured and fluid can be drawnfrom the blister. In another technique, an interstitial region can beaccessed and fluid drawn from that region without first forming a poolof fluid via a suction blister or the like. For example, a needle or amicroneedle can be applied to the interstitial region and fluid can bedrawn there from. In some cases, however, fluid may be withdrawn fromthe skin even without creating a suction blister or a pooled region offluid within the skin.

Where needles are used, it can be advantageous to select needles oflength such that interstitial fluid is preferentially obtained and,where not desirable, blood is not accessed. Those of ordinary skill inthe art can arrange needles relative to the skin for these purposesincluding, in one embodiment, introducing needles into the skin at anangle, relative to the skin's surface, other than 90°, i.e., tointroduce a needle or needles into the skin in a slanting fashion so asto limit the depth of penetration. In another embodiment, however, theneedles may enter the skin at approximately 90°.

Pooled regions of fluids, if present, may be formed on any suitablelocation within the skin of a subject. Factors such as safety orconvenience may be used to select a suitable location, as (in humans)the skin is relatively uniform through the body, with the exception ofthe hands and feet. As non-limiting examples, the pooled region may beformed on an arm or a leg, on the hands (e.g., on the back of the hand),on the feet, on the chest, abdomen, or the back of the subject, or thelike. The size of the pooled region of fluid that is formed in the skinand/or the duration that the pooled region lasts within the skin dependson a variety of factors, such as the technique of creating the pooledregion, the size of the pooled region, the size of the region of skin towhich the technique is applied, the amount of fluid removed from thepooled region (if any), any materials that are delivered into the pooledregion, or the like. For example, if vacuum is applied to the skin tocreate a suction blister, the vacuum applied to the skin, the durationof the vacuum, and/or the area of the skin affected may be controlled tocontrol the size and/or duration of the suction blister. In someembodiments, it may be desirable to keep the pooled regions relativelysmall, for instance, to prevent an unsightly visual appearance, to allowfor greater sampling accuracy (due to a smaller volume of material), orto allow for more controlled placement of particles within the skin. Forexample, the volume of the pooled region may be kept to less than about2 ml, less than about 1 ml, less than about 500 microliters, less thanabout 300 microliters, less than about 100 microliters, less than about50 microliters, less than about 30 microliters, less than about 10microliters, etc., in certain cases, or the average diameter of thepooled region (i.e., the diameter of a circle having the same area asthe pooled region) may be kept to less than about 5 cm, less than about4 cm, less than about 3 cm, less than about 2 cm, less than about 1 cm,less than about 5 mm, less than about 4 mm, less than about 3 mm, lessthan about 2 mm, or less than about 1 mm.

A variety of techniques may be used to cause pooled regions of fluid toform within the skin and/or to withdraw a bodily fluid from the skin ofa subject such as interstitial fluid or blood. In one set ofembodiments, vacuum is applied to create a suction blister, or otherwiseused to collect interstitial fluid or blood from a subject. In one setof embodiments, for example, a device containing a vacuum source, forinstance, a self-contained vacuum source such as a pre-packaged vacuumchamber, may be used to withdraw blood or interstitial fluid from thesubject. The fluid may be analyzed and/or stored for later use.

In other embodiments, other methods may be used to create as a pooledregion of fluid within the skin and/or withdraw fluid from the skinbesides, or in addition to, the use of vacuum. When vacuum (i.e., theamount of pressure below atmospheric pressure, such that atmosphericpressure has a vacuum of 0 mmHg, i.e., the pressure is gauge pressurerather than absolute pressure) is used to at least partially separatethe dermis from the epidermis to cause the pooled region to form, thepooled region of fluid thus formed can be referred to as a suctionblister. For example, pressures of at least about 50 mmHg, at leastabout 100 mmHg, at least about 150 mmHg, at least about 200 mmHg, atleast about 250 mmHg, at least about 300 mmHg, at least about 350 mmHg,at least about 400 mmHg, at least about 450 mmHg, at least about 500mmHg, at least about 550 mmHg, at least about 600 mmHg, at least about650 mmHg, at least about 700 mmHg, or at least about 750 mmHg may beapplied to the skin, e.g., to cause a suction blister and/or to collectinterstitial fluid from a subject (as discussed, these measurements arenegative relative to atmospheric pressure). For instance, a vacuumpressure of 100 mmHg corresponds to an absolute pressure of about 660mmHg (i.e., 100 mmHg below 1 atm). Different amounts of vacuum may beapplied to different subjects in some cases, for example, due todifferences in the physical characteristics of the skin of the subjects.

The vacuum may be applied to any suitable region of the skin, and thearea of the skin to which the vacuum may be controlled in some cases.For instance, the average diameter of the region to which vacuum isapplied may be kept to less than about 5 cm, less than about 4 cm, lessthan about 3 cm, less than about 2 cm, less than about 1 cm, less thanabout 5 mm, less than about 4 mm, less than about 3 mm, less than about2 mm, or less than about 1 mm. In addition, such vacuums may be appliedfor any suitable length of time at least sufficient to cause at leastsome separation of the dermis from the epidermis to occur. For instance,vacuum may be applied to the skin for at least about 1 min, at leastabout 3 min, at least about 5 min, at least about 10 min, at least about15 min, at least about 30 min, at least about 45 min, at least about 1hour, at least about 2 hours, at least about 3 hours, at least about 4hours, etc. Examples of devices suitable for creating such suctionblisters are discussed in more detail below. In other cases, however,bodily fluids such as blood or interstitial fluid may be removed fromthe skin using vacuum without the creation of a suction blister. Othernon-limiting fluids include saliva, sweat, tears, mucus, plasma, lymph,or the like.

Other methods besides vacuum may be used to cause such separation tooccur. For example, in another set of embodiments, heat may be used. Forinstance, a portion of the skin may be heated to at least about 40° C.,at least about 50° C., or at least about 55° C., using any suitabletechnique, to cause such separation to occur. In some (but not all)cases, the temperature may be limited to no more than about 60° C. or nomore than about 55° C. The skin may be heated, for instance, using anexternal heat source (e.g., radiant heat or a heated water bath), achemical reaction, electromagnetic radiation (e.g., microwave radiation,infrared radiation, etc.), or the like. In some cases, the radiation maybe focused on a relatively small region of the skin, e.g., to at leastpartially spatially contain the amount of heating within the skin thatoccurs.

In yet another set of embodiments, a separation chemical may be appliedto the skin to at least partially cause separation of the dermis and theepidermis to occur. Non-limiting examples of such separation chemicalsinclude proteases such as trypsin, purified human skin tryptase, orcompound 48/80. Separation compounds such as these are commerciallyavailable from various sources. The separation chemical may be applieddirectly to the skin, e.g., rubbed into the surface of the skin, or insome cases, the separation chemical can be delivered into the subject,for example, between the epidermis and dermis of the skin. Theseparation chemical can, for example, be injected in between the dermisand the epidermis.

Another example of a separation chemical is a blistering agent, such aspit viper venom or blister beetle venom. Non-limiting examples ofblistering agents include phosgene oxime, Lewisite, sulfur mustards(e.g., mustard gas or 1,5-dichloro-3-thiapentane,1,2-bis(2-chloroethylthio)ethane, 1,3-bis(2-chloroethylthio)-n-propane,1,4-bis(2-chloroethylthio)-n-butane,1,5-bis(2-chloroethylthio)-n-pentane, 2-chloroethylchloromethylsulfide,bis(2-chloroethyl)sulfide, bis(2-chloroethylthio)methane,bis(2-chloroethylthiomethyl)ether, or bis(2-chloroethylthioethyl)ether),or nitrogen mustards (e.g., bis(2-chloroethyl)ethylamine,bis(2-chloroethyl)methylamine, or tris(2-chloroethyl)amine).

In still another set of embodiments, a device may be inserted into theskin and used to mechanically separate the epidermis and the dermis, forexample, a wedge or a spike. Fluids may also be used to separate theepidermis and the dermis, in yet another set of embodiments. Forexample, saline or another relatively inert fluid may be injected intothe skin between the epidermis and the dermis to cause them to at leastpartially separate.

These and/or other techniques may also be combined, in still otherembodiments. For example, in one embodiment, vacuum and heat may beapplied to the skin of a subject, sequentially and/or simultaneously, tocause such separation to occur. As a specific example, in oneembodiment, vacuum is applied while the skin is heated to a temperatureof between about 40° C. and about 50° C.

The fluid contained within the skin, e.g., within the pooled region offluid is typically drawn from the surrounding dermal and/or epidermallayers within the skin, and includes interstitial fluid, or even bloodin some cases. In some cases, such fluids may be collected even withoutcreating a suction blister within the skin. For instance, a vacuum maybe applied to the skin, e.g., through a needle as described herein, towithdraw interstitial fluid from the skin.

Often, such fluids will contain various analytes within the body thatare important for diagnostic purposes, for example, markers for variousdisease states, such as glucose (e.g., for diabetics); other exampleanalytes include ions such as sodium, potassium, chloride, calcium,magnesium, and/or bicarbonate (e.g., to determine dehydration); gasessuch as carbon dioxide or oxygen; H⁺ (i.e., pH); metabolites such asurea, blood urea nitrogen or creatinine; hormones such as estradiol,estrone, progesterone, progestin, testosterone, androstenedione, etc.(e.g., to determine pregnancy, illicit drug use, or the like); orcholesterol. Other examples include insulin, or hormone levels.

As previously discussed, agents such as particles can be used to analyzebodily fluids, such as interstitial fluids. In some cases, the particlesmay be used to determine pH or metal ions, proteins, enzymes,antibodies, nucleic acids (e.g. DNA, RNA, etc.), drugs, sugars (e.g.,glucose), hormones (e.g., estradiol, estrone, progesterone, progestin,testosterone, androstenedione, etc.), carbohydrates, or other analytesof interest. Other conditions that can be detected can include pHchanges, which may indicate disease, yeast infection, periodontaldisease at a mucosal surface, oxygen or carbon monoxide levels whichindicate lung dysfunction, and drug levels, both legal prescriptionlevels of drugs such as coumadin and illegal such as cocaine ornicotine. Further examples of analytes include those indicative ofdisease, such as cancer specific markers such as CEA and PSA, viral andbacterial antigens, and autoimmune indicators such as antibodies todouble stranded DNA, indicative of Lupus. Still other conditions includeexposure to elevated carbon monoxide, which could be from an externalsource or due to sleep apnea, too much heat (important in the case ofbabies whose internal temperature controls are not fullyself-regulating) or from fever.

Still other potentially suitable analytes include various pathogens suchas bacteria or viruses, and/or markers produced by such pathogens. Thus,in certain embodiments of the invention, as discussed below, one or moreanalytes within the skin, e.g., within a pooled region of fluid, may bedetermined in some fashion, which may be useful in determining a past,present and/or future condition of the subject.

In one embodiment as discussed below, an analyte may be determined as an“on/off” or “normal/abnormal” situation. In some cases, the particles(or other agents) indicate a change. Detection of the analyte, forexample, may be indicative that insulin is needed; a trip to the doctorto check cholesterol; ovulation is occurring; kidney dialysis is needed;drug levels are present (e.g., especially in the case of illegal drugs)or too high/too low (e.g., important in care of geriatrics in particularin nursing homes). As another embodiment, however, an analyte may bedetermined quantitatively.

In some embodiments of the invention, one or more materials may bedelivered to the skin. Examples of suitable materials include, but arenot limited to, particles such as microparticles or nanoparticles, achemical, a drug or a therapeutic agent, a diagnostic agent, a carrier,or the like. The materials may be delivered into the skin using anysuitable technique; various techniques for delivery into the skin arewell-known to those of ordinary skill in the art. Examples of suitabledelivery techniques include, but are not limited to, injection (e.g.,using needles such as hypodermic needles) or a jet injector, such asthose discussed below.

In one set of embodiments, particles are delivered to the skin. Theparticles may be, for example, nanoparticles or microparticles, and insome cases, the particles may be anisotropic particles. Examples of suchparticles are discussed in more detail herein. In some cases, aplurality of particles may be used, and in some cases, some, orsubstantially all, of the particles may be the same. For example, atleast about 10%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, or at least about 99% of theparticles may have the same shape, and/or may have the same composition.For example, in one embodiment, at least about 50% of the particlesdelivered to the skin may have the same shape, and/or may have the samecomposition. For instance, at least about 50% of the particles may beanisotropic particles.

The particles may be used for a variety of purposes. For instance, theparticles may contain a diagnostic agent or a reaction entity able tointeract with and/or associate with an analyte, or another reactionentity, or other particles. Such particles may be useful, for example,to determine one or more analytes, such as a marker of a disease state,as discussed below. As another example, the particles may contain a drugor a therapeutic agent, positioned on the surface and/or internally ofthe particles, which may be released by the particles and delivered tothe subject. Specific examples of these and other embodiments arediscussed in detail below.

In some cases, materials such as particles may become embedded withinthe skin, e.g., within a pooled region of fluid, for example, due tophysical properties of the materials (e.g., size, hydrophobicity, etc.),and/or by draining at least a portion of the fluid within the pooledregion such that the material is unable to escape, thereby remainingcontained between the dermis and epidermis layers of the skin. Thus, insome cases, a depot of material may be formed within the skin, and thedepot may be temporary or permanent. For instance, materials within thedepot may eventually degrade (e.g., if the material biodegradable),enter the bloodstream, or be sloughed off to the environment, e.g., asthe cells of the dermis differentiate to form new epidermis andaccordingly push the material towards the surface of the skin. Thus, thedepot of material may be present within the subject on a temporary basis(e.g., on a time scale of days or weeks), in certain instances.

Fluid may be drained from the pooled region using any suitabletechnique, for example, by externally removing the fluid from the pooledregion (e.g., using techniques such as those discussed below), and/or byremoving the vacuum or other stimulus used to cause the pooled region tooccur, thereby allowing the fluid to become resorbed within the subject.Such resorption may occur, for example on a time scale of minutes tohours, depending on factors such as the size or volume of the pooledregion of fluid. For example, in one embodiment, fluid is withdrawnusing a needle such as a hypodermic needle. In some cases, this needlemay also be used to deliver particles or other materials to the skin.

Fluids may also be externally removed from skin, e.g., from a pooledregion of fluid, for example, to at least partially drain the pooledregion of fluid, and/or for analysis. For instance, at least a portionof the fluid may be stored, and/or analyzed to determine one or moreanalytes, e.g., a marker for a disease state, or the like. The fluidwithdrawn from the skin may be subjected to such uses, and/or one ormore materials previously delivered to the skin (e.g., particles) may besubject to such uses. The fluid may be removed using any suitabletechnique. For example, in one embodiment, fluid is withdrawn using aneedle such as a hypodermic needle. In some cases, this needle may alsobe used to deliver particles or other materials to the skin. The fluidmay also be withdrawn using vacuums such as those discussed herein inanother embodiment of the invention. For example, vacuum may be appliedto a conduit, such as a needle, in fluidic communication withinterstitial fluid, e.g., within a pooled region of fluid, in order todraw up at least a portion of the fluid from the pooled region. In yetanother embodiment, fluid is withdrawn using capillary action (e.g.,using a hypodermic needle having a suitably narrow inner diameter). Instill another embodiment, pressure may be applied to force fluid out ofthe needle.

In still another embodiment, fluid may be withdrawn using a hygroscopicagent applied to the surface of the skin, or proximate the skin. In somecases, pressure may be applied to drive the hygroscopic agent into theskin. Hygroscopic agents typically are able to attract water from thesurrounding environment, for instance, through absorption or adsorption.Non-limiting examples of hygroscopic agents include sugar, honey,glycerol, ethanol, methanol, sulfuric acid, methamphetamine, iodine,many chloride and hydroxide salts, and a variety of other substances.Other examples include, but are not limited to, zinc chloride, calciumchloride, potassium hydroxide or sodium hydroxide. In some cases, asuitable hygroscopic agent may be chosen based on its physical orreactive properties, e.g., inertness or biocompatibility towards theskin of the subject, depending on the application.

In some cases, fluids or other materials delivered to the subject may beused for indication of a past, present and/or future condition of thesubject. Thus, the condition of the subject to be determined may be onethat is currently existing in the subject, and/or one that is notcurrently existing, but the subject is susceptible or otherwise is at anincreased risk to that condition. The condition may be a medicalcondition, e.g., diabetes or cancer, or other physiological conditions,such as dehydration, pregnancy, illicit drug use, or the like.Additional non-limiting examples are discussed below. In one set ofembodiments, the materials may include a diagnostic agent, for example,one which can determine an analyte within the subject, e.g., one that isa marker for a disease state. Examples of such markers have beendiscussed above. As a specific non-limiting example, material deliveredto the skin, e.g., to the dermis or epidermis, to a pooled region offluid, etc., of a subject may include a particle including an antibodydirected at a marker produced by bacteria.

In other cases, however, the materials delivered to the subject may beused to determine conditions that are external to the subject. Forexample, the materials may contain reaction entities able to recognizepathogens or other environmental conditions surrounding the subject, forexample, an antibody able to recognize an external pathogen (or pathogenmarker). As a specific example, the pathogen may be anthrax and theantibody may be an antibody to anthrax spores. As another example, thepathogen may be a Plasmodia (some species of which causes malaria) andthe antibody may be an antibody that recognizes the Plasmodia.

Another aspect of the present invention is generally directed to devicesable to cause the formation of the pooled region of fluids within theskin of a subject, and in some cases, to devices able to deliver and/orremove fluids or other materials from the pooled region of fluids. Itshould be understood, however, that other devices in other aspects donot require the formation of pooled regions of fluids within the skin.In some cases, the device may be able to collect bodily fluids such asinterstitial fluid or blood from the skin, including fluid from a pooledregion of fluid, or from other locations. For example, the device maytake the form of a skin “patch,” according to one embodiment. Typically,a skin patch includes one or more layers of material that are adhered tothe surface of the skin, and can be applied by the subject or anotherperson. In certain embodiments, layers or portions of the skin patch maybe removed, leaving other layers or portions behind on the skin. Often,the skin patch lacks an external power source, although the variouslayers of the patch may contain various chemicals, such as drugs,therapeutic agents, diagnostic agents, reaction entities, etc. In somecases, the skin patch may also include mechanical elements as well, forexample, a cutter such as is discussed herein.

As a specific, non-limiting example, in one embodiment, a skin patch orother device may be used to create a suction blister without an externalpower and/or a vacuum source. Examples of such devices include, besidesskin patches, strips, tapes, bandages, or the like. For instance, a skinpatch may be contacted with the skin of a subject, and a vacuum createdthrough a change in shape of a portion of the skin patch or other device(e.g., using a shape memory polymer), which may be used to create asuction blister and/or withdraw fluid from the skin. As a specificexample, a shape memory polymer may be shaped to be flat at a firsttemperature (e.g., room temperature) but curved at a second temperature(e.g., body temperature), and when applied to the skin, the shape memorypolymer may alter from a flat shape to a curved shape, thereby creatinga vacuum. As another example, a mechanical device may be used to createthe vacuum, For example, springs, coils, expanding foam (e.g., from acompressed state), a shape memory polymer, shape memory metal, or thelike may be stored in a compressed or wound released upon application toa subject, then released (e.g., unwinding, uncompressing, etc.), tomechanically create the vacuum. Thus, in some cases, the device is“pre-packaged” with a suitable vacuum source (e.g., a pre-evacuatedvacuum chamber); for instance, in one embodiment, the device may beapplied to the skin and activated in some fashion to create and/oraccess the vacuum source. One example is described below with respect toFIG. 8. In yet another example, a chemical reaction may be used tocreate a vacuum, e.g., a reaction in which a gas is produced, which canbe harnessed to provide the mechanical force to create a vacuum. Instill another example, a component of the device may be able to create avacuum in the absence of mechanical force. In another example, thedevice may include a self-contained vacuum actuator, for example,chemical reactants, a deformable structure, a spring, a piston, etc.

FIGS. 8C-8E illustrates an embodiment where a compressed foam is used tocreate a suction blister. In FIG. 8C, device 80 is placed on skin 57,and includes compressed foam 95. Device 80 includes a cutter 54 able tocut a portion of the skin, e.g., when pressed down onto the skin as isshown in FIG. 8D, e.g., creating a hole 97. In addition, the foam may beallowed to expand in some fashion after the device has been placed onthe skin. For example, a housing portion of device 80 may be removed toallow expansion of the foam to occur. Expansion of the foam, as is shownin FIG. 8E, may create a suction and thereby cause the formation of asuction blister 99, and/or may allow fluids to be withdrawn from theskin.

Accordingly, in one set of embodiments, the skin patch or other devicemay be used to create a suction blister automatically, once activated,without any external control by a user. In other embodiments, however,the device may be larger. For instance, the device may be a handhelddevice that is applied to the surface of the skin of a subject. In somecases, however, the device may be sufficiently small or portable thatthe subject can self-administer the device. In certain embodiments, thedevice may also be powered. In some instances, the device may be appliedto the surface of the skin, and is not inserted into the skin.

In other embodiments, however, at least a portion of the device may beinserted into the skin, for example, mechanically. For example, in oneembodiment, the device may include a cutter, such as a hypodermicneedle, a knife blade, a piercing element (e.g., a solid or hollowneedle), or the like, as discussed herein. In some cases, the device maycomprise a cutter able to cut or pierce the surface of the skin. Thecutter may comprise any mechanism able to create a path to a fluidwithin the skin, e.g., through which fluids may be delivered and/orremoved from the skin. For example, the cutter may comprise a hypodermicneedle, a knife blade, a piercing element (e.g., a solid or a hollowneedle), or the like, which can be applied to the skin to create asuitable conduit for the withdrawal of fluid from the skin. In oneembodiment, a cutter is used to create such a pathway and removed, thenfluid is removed via this pathway using any suitable technique. Inanother embodiment, the cutter remains in place within the skin, andfluid may be drawn through a conduit within the cutter.

As an example, the device may be constructed such that a cutter or aneedle is inserted into the skin after a suction blister is formed. Insome cases, the device may be designed such that portions of the deviceare separable. For example, a first portion of the device may be removedfrom the surface of the skin, leaving other portions of the devicebehind on the skin. In one embodiment, a stop may also be included toprevent or control the depth to which the cutter or other device insertsinto the skin, e.g., to control penetration to the epidermis, dermis,etc.

Various non-limiting examples of such devices are illustrated in FIG. 2.In FIG. 2A, device 50 includes an interface 25 that can be used to applyvacuum to the surface of the skin 10, thereby creating a pooled regionof fluid 20 within the skin between the epidermis 15 and the dermis 17.In the embodiment illustrated, device 50 includes conduit 27, which canbe connected in fluidic communication with a vacuum source, such as avacuum pump or an external (line) vacuum source. In this figure, device50 also includes a cutter, in this case a hypodermic needle, that can beextended into the pooled region of fluid, and used to deliver and/orremove fluids or other materials from the pooled region of fluid. Inthis figure, the hypodermic needle is used both to create a conduitwithin the skin and to delivery and/or remove fluids from the skin; inother embodiments, however, the needle (or other device) may be removedfrom the skin, leaving behind a “hole” within the skin through whichfluids can be delivered and/or removed. In this figure, fluid withdrawnfrom the pooled region of fluid may be delivered to a sensor 52, whichcan be used to determine an analyte, such as a marker for a diseasestate, that is present within skin 10. The cutter may be an integralpart of device 50, or a separate device. In some cases, device 50 maycomprise separably removable portions. For instance, after creating thesuction blister, interface 25 may be removable from device 50, leavinghypodermic needle 40 within skin 10.

Another example of a device is shown in FIG. 2B. In this figure, device10 includes conduit 27, in which fluids can be delivered and/or removed.In one embodiment, a fluid, such as saline, is delivered through conduit27, thereby creating a pooled region of fluid between the epidermis 15and the dermis 17. Optionally, materials such as particles may bedelivered into the pooled region of fluid using conduit 27, e.g., fortherapeutic or diagnostic purposes. In some cases, after creation of thepooled region of fluid within skin 10, at least a portion of the fluidmay be removed through conduit 27 (or a separate conduit, in some cases)for analysis, e.g., as previously discussed.

In FIG. 2C, a similar device is shown, including a cutter 54, such as ahypodermic needle or a knife blade, that can be inserted into the skinand used to at least partially separate the epidermis from the dermis toallow for the creation of a pooled region of fluid 20. For example,cutter 54 may separate the epidermis from the dermis by delivering aseparation chemical to this region, and/or by conducting heat to thisregion. Also shown in this example is conduit 27, which may be used todeliver and/or remove fluid from the pooled region of fluid. In somecases, cutter 54 and conduit 27 may be separately administered to thesubject, e.g., sequentially or simultaneously. In another embodiment,both may be part of the same device that is administered to skin 10, andin some cases, cutter 54 may be separated from conduit 27, e.g., suchthat cutter 54 can be removed from the skin while conduit 27 remainswithin the skin.

FIG. 3A illustrates a device according to another embodiment of theinvention. In this example, a device 60 is applied to skin 63. Device 60may be, for instance, a patch, an appliqué, a mechanical device, or thelike. A vacuum is created between device 60 and the skin in region 65.The vacuum may be contained by the device itself, and/or throughconnection with a vacuum source, such as a vacuum pump or an external(line) vacuum source. Other examples of vacuum sources include, but arenot limited to, syringes, bulbs, vacuum pumps, Venturi tubes, or evenmanual (mouth) suction. Also shown in FIG. 3B is cutter 70. Cutter 70may be, for example, a needle or a microneedle, a knife blade, or thelike.

As the suction blister forms within the skin, portions of the skin maybe uplifted due to the pooled region of fluid, shown in FIG. 3B asportion 64 extending upwardly into region 65 of device 60. As portion 64extends upward into region 65, it comes into contact with cutter 70.Under certain conditions, the skin may extend upward sufficiently intoregion 65 that cutter 70 cuts into skin 65, as is shown in FIG. 3C. Forinstance, if cutter 70 is a hypodermic needle, upon piercing of theneedle into the skin, the needle may be used to access interstitialfluid within the skin, e.g., within a pooled region of fluid within theskin forming the suction blister. Accordingly, fluid may be withdrawnand/or materials such as particles may be delivered into the pooledregion using the hypodermic needle.

As additional examples, the device may comprise a first portion able tocreate a pooled region of fluid within the skin of a subject and asecond portion able to determine fluid removed from the pooled region,or a first portion able to create a pooled region of fluid within theskin of a subject and a second portion able to deliver fluid to thepooled region of fluid, where the various portions may be separated fromeach other. For instance, the fluid itself may be determined (forexample, the presence and/or absence of the fluid, the concentration offluid, the volume of fluid, etc.), or an analyte within the fluid may bedetermined, e.g., qualitatively or quantitatively, whether the analyteis present and/or absent, etc. As yet another example, the device maycomprise a first portion able to create a pooled region of fluid withinthe skin of a subject, a second portion able to determine fluid removedfrom the pooled region, and a third portion able to deliver fluid to thepooled region of fluid, where some or all of the portions may beseparated from each other.

In certain embodiments, the device is able to create a pooled region offluid within the skin of a subject. In one embodiment, the device isable to create vacuum on the surface of the skin, e.g., to form asuction blister within the skin. In other embodiments, however, thedevice may create a vacuum to withdraw fluid from the skin withoutnecessarily creating a pooled region of fluid or forming a suctionblister within the skin. In one set of embodiments, fluids may bedelivered to or withdrawn from the skin using vacuum. The vacuum may bean external vacuum source, and/or the vacuum source may beself-contained within the device. For example, vacuums of at least about50 mmHg, at least about 100 mmHg, at least about 150 mmHg, at leastabout 200 mmHg, at least about 250 mmHg, at least about 300 mmHg, atleast about 350 mmHg, at least about 400 mmHg, at least about 450 mmHg,at least about 500 mmHg, at least 550 mmHg, at least 600 mmHg, at least650 mmHg, at least about 700 mmHg, or at least about 750 mmHg may beapplied to the skin to cause a suction blister. Any source of vacuum maybe used. For example, the device may comprise a vacuum source, and/or beconnectable to a vacuum source is external to the device, such as avacuum pump or an external (line) vacuum source. In some cases, vacuummay be created manually, e.g., by manipulating a syringe pump or thelike, or the low pressure may be created mechanically or automatically,e.g., using a piston pump, a syringe, a bulb, a Venturi tube, manual(mouth) suction, etc. or the like.

As mentioned, any source of vacuum may be used. For example, the devicemay comprise an internal vacuum source, and/or be connectable to avacuum source is external to the device, such as a vacuum pump or anexternal (line) vacuum source.

In one set of embodiments, a device of the present invention may nothave an external power and/or a vacuum source. In some cases, the deviceis “pre-loaded” with a suitable vacuum source; for instance, in oneembodiment, the device may be applied to the skin and activated in somefashion to create and/or access the vacuum source. As one example, adevice of the present invention may be contacted with the skin of asubject, and a vacuum created through a change in shape of a portion ofthe device (e.g., using a shape memory polymer), or the device maycontain one or more sealed, self-contained vacuum compartments, where aseal is punctured in some manner to create a vacuum. For instance, uponpuncturing the seal, a vacuum compartment may be in fluidiccommunication with a needle, which can be used to move the skin towardsthe device, withdraw fluid from the skin, or the like.

As another example, a shape memory polymer may be shaped to be flat at afirst temperature (e.g., room temperature) but curved at a secondtemperature (e.g., body temperature), and when applied to the skin, theshape memory polymer may alter from a flat shape to a curved shape,thereby creating a vacuum. As yet another example, a mechanical devicemay be used to create the vacuum, For example, springs, coils, expandingfoam (e.g., from a compressed state), a shape memory polymer, shapememory metal, or the like may be stored in a compressed or woundreleased upon application to a subject, then released (e.g., unwinding,uncompressing, etc.), to mechanically create the vacuum. Non-limitingexamples of shape-memory polymers and metals include Nitinol,compositions of oligo(epsilon-caprolactone)diol and crystallizableoligo(rho-dioxanone)diol, or compositions ofoligo(epsilon-caprolactone)dimethacrylate and n-butyl acrylate.

In some cases, the device includes an interface that is able to applyvacuum to the skin. The interface may be, for example, a suction cup ora circular bowl that is placed on the surface of the skin, and vacuumapplied to the interface to create a vacuum. In one set of embodiments,the interface is part of a support structure, as discussed herein. Theinterface may be formed from any suitable material, e.g., glass, rubber,polymers such as silicone, polyurethane, nitrile rubber, EPDM rubber,neoprene, or the like. In some cases, the seal between the interface andthe skin may be enhanced (e.g., reducing leakage), for instance, usingvacuum grease, petroleum jelly, a gel, a hydrogel, or the like. In somecases, the interface may be relatively small, for example, having adiameter of less than about 5 cm, less than about 4 cm, less than about3 cm, less than about 2 cm, less than about 1 cm, less than about 5 mm,less than about 4 mm, less than about 3 mm, less than about 2 mm, orless than about 1 mm. The interface may be circular, although othershapes are also possible, for example, square, star-shaped (having 5, 6,7, 8, 9, 10, 11, etc. points), tear-drop, oval, rectangular, or thelike. In some cases, non-circular shapes may be used since high-energypoints, e.g., the points or corners of the shape may enhance oraccelerate blister formation. Non-limiting examples of such shapes areshown in FIG. 6. Other non-circular shapes besides these may also beused in other embodiments.

The interface may also be selected, in some cases, to keep the size ofthe pooled region below a certain area, e.g., to minimize pain ordiscomfort to the subject, for aesthetic reasons, or the like. Theinterface may be constructed out of any suitable material, e.g., glass,plastic, or the like.

The device may also comprise, in some cases, a portion able to delivermaterials such as particles into the skin, for example, into the dermisor epidermis, into a pooled region within the skin, etc. For example,the device may include a needle such as a hypodermic needle ormicroneedles, or jet injectors such as those discussed below. As anexample, in one embodiment, a needle such as a hypodermic needle can beused to deliver and/or withdraw fluid to or from the skin. In somecases, for example, fluid may be delivered and/or withdrawn from apooled region of fluid in the skin, if present. Hypodermic needles arewell-known to those of ordinary skill in the art, and can be obtainedcommercially with a range of needle gauges. For example, the needle maybe in the 20-30 gauge range, or the needle may be 32 gauge, 33 gauge, 34gauge, etc.

Accordingly, in one set of embodiments, many techniques for deliveringand/or withdrawing fluid are described in the applications incorporatedherein. It is to be understood that, generally, fluids may be deliveredand/or withdrawn in a variety of ways, and various systems and methodsfor delivering and/or withdrawing fluid from the skin are discussedherein. It should also be understood that techniques for deliveringmaterials into a pooled region of the skin are by way of example only,and that in other aspects, the invention is directed to techniques fordelivering and/or withdrawing fluid from the skin of a subject (with orwithout the presence of a pooled region of the skin). Some additionalnon-limiting examples of such techniques are discussed below. In one setof embodiments, for example, techniques for piercing or altering thesurface of the skin to transport a fluid are discussed, for example, aneedle such as a hypodermic needle or microneedles, chemicals applied tothe skin (e.g., penetration enhancers), or jet injectors or othertechniques such as those discussed below.

If needles are present, the needles may be of any suitable size andlength, and may be solid or hollow. The needles may have any suitablecross-section (e.g., perpendicular to the direction of penetration), forexample, circular, square, oval, elliptical, rectangular, roundedrectangle, triangular, polygonal, hexagonal, irregular, etc. Forexample, the needle may have a length of less than about 5 mm, less thanabout 4 mm, less than about 3 mm, less than about 2 mm, less than about1 mm, less than about 800 micrometers, less than 600 micrometers, lessthan 500 micrometers, less than 400 micrometers, less than about 300micrometers, less than about 200 micrometers, less than about 175micrometers, less than about 150 micrometers, less than about 125micrometers, less than about 100 micrometers, less than about 75micrometers, less than about 50 micrometers, etc. The needle may alsohave a largest cross-sectional dimension of less than about 5 mm, lessthan about 4 mm, less than about 3 mm, less than about 2 mm, less thanabout 1 mm, less than about 800 micrometers, less than 600 micrometers,less than 500 micrometers, less than 400 micrometers, less than about300 micrometers, less than about 200 micrometers, less than about 175micrometers, less than about 150 micrometers, less than about 125micrometers, less than about 100 micrometers, less than about 75micrometers, less than about 50 micrometers, etc. For example, in oneembodiment, the needle may have a rectangular cross section havingdimensions of 175 micrometers by 50 micrometers. In one set ofembodiments, the needle may have an aspect ratio of length to largestcross-sectional dimension of at least about 2:1, at least about 3:1, atleast about 4:1, at least 5:1, at least about 7:1, at least about 10:1,at least about 15:1, at least about 20:1, at least about 25:1, at leastabout 30:1, etc. In one embodiment, the needle is a microneedle.

For example, the needle may be a microneedle such as those disclosed inU.S. Pat. No. 6,334,856, issued Jan. 1, 2002, entitled “MicroneedleDevices and Methods of Manufacture and Use Thereof,” by Allen, et al.,and the microneedle may be used to deliver and/or withdraw fluids orother materials to or from the skin of a subject. The microneedles maybe hollow or solid, and may be formed from any suitable material, e.g.,metals, ceramics, semiconductors, organics, polymers, and/or composites.Examples include, but are not limited to, pharmaceutical grade stainlesssteel, titanium, nickel, iron, gold, tin, chromium, copper, alloys ofthese or other metals, silicon, silicon dioxide, and polymers, includingpolymers of hydroxy acids such as lactic acid and glycolic acidpolylactide, polyglycolide, polylactide-co-glycolide, and copolymerswith polyethylene glycol, polyanhydrides, polyorthoesters,polyurethanes, polybutyric acid, polyvaleric acid,polylactide-co-caprolactone, polycarbonate, polymethacrylic acid,polyethylenevinyl acetate, polytetrafluorethylene, polymethylmethacrylate, polyacrylic acid, or polyesters. In some cases, more thanone microneedle may be used. For example, arrays of microneedles may beused, and the microneedles may be arranged in the array in any suitableconfiguration, e.g., periodic, random, etc. In some cases, the array mayhave 3 or more, 4 or more, 5 or more, 6 or more, 10 or more, 15 or more,20 or more, 35 or more, 50 or more, 100 or more, or any other suitablenumber of microneedles. It should be understood that references to“needle” or “microneedle” as discussed herein are by way of example andease of presentation only, and that in other embodiments, more than oneneedle and/or microneedle may be present in any of the descriptionsherein.

As still another example, pressurized fluids may be used to deliverfluids or other materials into the skin, for instance, using a jetinjector or a “hypospray.” Typically, such devices produce ahigh-pressure “jet” of liquid or powder (e.g., a biocompatible liquid,such as saline) that drives material into the skin, and the depth ofpenetration may be controlled, for instance, by controlling the pressureof the jet. The pressure may come from any suitable source, e.g., astandard gas cylinder or a gas cartridge. A non-limiting example of sucha device can be seen in U.S. Pat. No. 4,103,684, issued Aug. 1, 1978,entitled “Hydraulically Powered Hypodermic Injector with Adapters forReducing and Increasing Fluid Injection Force,” by Ismach.Pressurization of the liquid may be achieved, for example, usingcompressed air or gas, for instance, from a gas cylinder or a gascartridge.

In addition, in certain embodiments, the device may comprise a portionable to remove at least a portion of the fluid from the skin. Forinstance, the device may comprise a hypodermic needle, a vacuum source,a hygroscopic agent, or the like. In certain cases, the portion of thedevice able to remove fluid may also be used to deliver fluids to theskin. Fluid may be removed from the skin using any suitable technique.For instance, in one embodiment, the fluid is removed manually, e.g., bymanipulating a plunger on a syringe. In another embodiment, the fluidcan be removed from the skin mechanically or automatically, e.g., usinga piston pump or the like.

In some aspects, the device may include channels such as microfluidicchannels, which may be used to deliver and/or withdraw fluids and/orother materials such as particles into or out of the skin, e.g., withinthe pooled region of fluid. In some cases, the microfluidic channels arein fluid communication with a fluid transporter that is used to deliverand/or withdraw fluids to or from the skin. For example, in one set ofembodiments, the device may include a hypodermic needle that can beinserted into the skin, and fluid may be delivered into the skin via theneedle and/or withdrawn from the skin via the needle. The device mayalso include one or more microfluidic channels to contain fluid fordelivery to the needle, e.g., from a source of fluid, and/or to withdrawfluid from the skin, e.g., for delivery to an analytical compartmentwithin the device, to a reservoir for later analysis, or the like.

One embodiment of a device including microfluidic channels is nowdisclosed with reference to FIG. 4A. In this figure, device 80,applicable to skin 87, includes a first compartment 81 and a secondcompartment 82. It should be noted that, in some embodiments, thecompartments are not necessarily discrete chambers as are shown in FIG.4A, and various compartments may be separated from each other throughthe use of controllers such as valves, membranes, or the like, e.g., asis shown in FIG. 7 with compartments 81, 82 and 83 and valves 91 and 92.Referring again to FIG. 4A, device 80 may be, for instance, a patch, anappliqué, a mechanical device, etc. First compartment 81 and secondcompartment 82 may be fluidic communication, e.g., using a microfluidicchannel 85. Optionally, a check valve 88 may be used to prevent backflowof fluid from second compartment 82 into first compartment 81 (or viceversa, depending on the embodiment). Check valve 88, if present, may beany valve that preferentially allows fluid flow in one direction,relative to the opposite direction. For instance, as is shown in FIG. 5,check valve may comprise one or more hinged portions that are able toswing in one direction (e.g., downstream), but are not able to swing inthe opposite direction (e.g., upstream).

In one example, vacuum is applied to the device from a vacuum sourceattached to channel 89, and the vacuum can be used to create a suctionblister in the skin, as discussed above. For example, first compartment81 may include a needle 86 that is used to access the fluid within theskin, e.g., within a pooled region of fluid created by the suctionblister. Second compartment 82 may be used, for example, to analyze acomponent of a fluid withdrawn from the skin. In another example,channel 89 may be connected to a source of pressure, and secondcompartment 82 may contain a fluid to be delivered to the skin.

In some cases, more than one compartment may be present within thedevice, and in some cases, some or all of the compartments may be influidic communication, e.g., via channels such as microfluidic channels.In various embodiments, a variety of compartments and/or channels may bepresent within the device, depending on the application. For example,the device may contain compartments for sensing an analyte, compartmentsfor holding reagents, compartments for controlling temperature,compartments for controlling pH or other conditions, compartments forcreating or buffering pressure or vacuum, compartments for controllingor dampening fluid flow, mixing compartments, or the like.

As a specific non-limiting example, in FIG. 4B, third compartment 83 isused to facilitate the creation of pressure or vacuum within firstcompartment 81. The compartments may be arranged in any suitablearrangement, e.g., as shown in FIG. 4B with first compartment 81 influid communication via a first channel to second compartment 82, whichis in fluid communication via a third channel to third compartment 83.As discussed above, first compartment 81 may be used to create a vacuumon the skin and/or access fluid within the skin, facilitated by vacuumcreated using third compartment 83, and fluid drawn from the skin maypass through microfluidic channel 85 (and optional check valve 88) toenter second compartment 82 for analysis. The analysis of the fluid maybe performed using any suitable technique such as those describedherein. For example, second compartment 82 may contain an agent able todetermine an analyte, e.g., particles producing a color change which isproportional to the amount of analyte.

Yet another example is described with reference to FIG. 8, showing top(FIG. 8A) and side (FIG. 8B) views of an example device 80. In thisfigure, first compartment 81 and second compartment 82 are in fluidiccommunication, e.g., using a microfluidic channel 85. A valve 88separates these compartments. The device may be “pre-loaded” with avacuum within second compartment 82, which is not in fluidiccommunication with first compartment due to closure of valve 88. Afterdevice 80 is applied to the skin 87 of a subject, e.g., in an air-tightfashion such that first compartment 81 is not exposed to the externalenvironment, valve 88 may be opened, thereby allowing first compartment81 to become fluidically exposed to the vacuum within second compartment82. In this way, vacuum may be applied to the skin via first compartment81 once valve 88 has been opened. Thus, certain embodiments of thepresent invention are directed to devices able to withdraw a fluid fromthe skin, e.g., blood or interstitial fluid, for analysis and/or storagefor later use.

Thus, in one set of embodiments, the device may include a microfluidicchannel. As used herein, “microfluidic,” “microscopic,” “microscale,”the “micro-” prefix (for example, as in “microchannel”), and the likegenerally refers to elements or articles having widths or diameters ofless than about 1 mm, and less than about 100 microns (micrometers) insome cases. In some embodiments, larger channels may be used instead of,or in conjunction with, microfluidic channels for any of the embodimentsdiscussed herein. For examples, channels having widths or diameters ofless than about 10 mm, less than about 9 mm, less than about 8 mm, lessthan about 7 mm, less than about 6 mm, less than about 5 mm, less thanabout 4 mm, less than about 3 mm, or less than about 2 mm may be used incertain instances. In some cases, the element or article includes achannel through which a fluid can flow. In all embodiments, specifiedwidths can be a smallest width (i.e. a width as specified where, at thatlocation, the article can have a larger width in a different dimension),or a largest width (i.e. where, at that location, the article has awidth that is no wider than as specified, but can have a length that isgreater). Thus, for instance, the microfluidic channel may have anaverage cross-sectional dimension (e.g., perpendicular to the directionof flow of fluid in the microfluidic channel) of less than about 1 mm,less than about 500 microns, less than about 300 microns, or less thanabout 100 microns. In some cases, the microfluidic channel may have anaverage diameter of less than about 60 microns, less than about 50microns, less than about 40 microns, less than about 30 microns, lessthan about 25 microns, less than about 10 microns, less than about 5microns, less than about 3 microns, or less than about 1 micron.

A “channel,” as used herein, means a feature on or in an article (e.g.,a substrate) that at least partially directs the flow of a fluid. Insome cases, the channel may be formed, at least in part, by a singlecomponent, e.g. an etched substrate or molded unit. The channel can haveany cross-sectional shape, for example, circular, oval, triangular,irregular, square or rectangular (having any aspect ratio), or the like,and can be covered or uncovered (i.e., open to the external environmentsurrounding the channel). In embodiments where the channel is completelycovered, at least one portion of the channel can have a cross-sectionthat is completely enclosed, and/or the entire channel may be completelyenclosed along its entire length with the exception of its inlet andoutlet.

A channel may have any aspect ratio, e.g., an aspect ratio (length toaverage cross-sectional dimension) of at least about 1:1, at least about2:1, more typically at least about 3:1, at least about 5:1, at leastabout 10:1, etc. As used herein, a “cross-sectional dimension,” inreference to a fluidic or microfluidic channel, is measured in adirection generally perpendicular to fluid flow within the channel. Achannel generally will include characteristics that facilitate controlover fluid transport, e.g., structural characteristics and/or physicalor chemical characteristics (hydrophobicity vs. hydrophilicity) and/orother characteristics that can exert a force (e.g., a containing force)on a fluid. The fluid within the channel may partially or completelyfill the channel. In some cases the fluid may be held or confined withinthe channel or a portion of the channel in some fashion, for example,using surface tension (e.g., such that the fluid is held within thechannel within a meniscus, such as a concave or convex meniscus). In anarticle or substrate, some (or all) of the channels may be of aparticular size or less, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm, less than about 2mm, less than about 1 mm, less than about 500 microns, less than about200 microns, less than about 100 microns, less than about 60 microns,less than about 50 microns, less than about 40 microns, less than about30 microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nmor less in some cases. In one embodiment, the channel is a capillary.

In some cases, the device may contain one or more chambers or reservoirsfor holding fluid. In some cases, the chambers may be in fluidiccommunication with one or more fluid transporters and/or one or moremicrofluidic channels. For instance, the device may contain a chamberfor collecting fluid withdrawn from a subject (e.g., for storage and/orlater analysis), a chamber for containing a fluid for delivery to thesubject (e.g., blood, saline, optionally containing drugs, hormones,vitamins, pharmaceutical agents, or the like), etc.

A variety of materials and methods, according to certain aspects of theinvention, can be used to form the device, e.g., microfluidic channels.For example, various components of the invention can be formed fromsolid materials, in which the channels can be formed via micromachining,film deposition processes such as spin coating and chemical vapordeposition, laser fabrication, photolithographic techniques, etchingmethods including wet chemical or plasma processes, and the like. See,for example, Scientific American, 248:44-55, 1983 (Angell, et al).

In one set of embodiments, various components of the systems and devicesof the invention can be formed of a polymer, for example, an elastomericpolymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene(“PTFE” or Teflon®), or the like. For instance, according to oneembodiment, a microfluidic channel may be implemented by fabricating thefluidic system separately using PDMS or other soft lithographytechniques (details of soft lithography techniques suitable for thisembodiment are discussed in the references entitled “Soft Lithography,”by Younan Xia and George M. Whitesides, published in the Annual Reviewof Material Science, 1998, Vol. 28, pages 153-184, and “Soft Lithographyin Biology and Biochemistry,” by George M. Whitesides, Emanuele Ostuni,Shuichi Takayama, Xingyu Jiang and Donald E. Ingber, published in theAnnual Review of Biomedical Engineering, 2001, Vol. 3, pages 335-373;each of these references is incorporated herein by reference).

Other examples of potentially suitable polymers include, but are notlimited to, polyethylene terephthalate (PET), polyacrylate,polymethacrylate, polycarbonate, polystyrene, polyethylene,polypropylene, polyvinylchloride, polytetrafluoroethylene, a fluorinatedpolymer, a silicone such as polydimethylsiloxane, polyvinylidenechloride, bis-benzocyclobutene (“BCB”), a polyimide, a fluorinatedderivative of a polyimide, or the like. Combinations, copolymers, orblends involving polymers including those described above are alsoenvisioned. The device may also be formed from composite materials, forexample, a composite of a polymer and a semiconductor material.

In some embodiments, various components of the invention are fabricatedfrom polymeric and/or flexible and/or elastomeric materials, and can beconveniently formed of a hardenable fluid, facilitating fabrication viamolding (e.g. replica molding, injection molding, cast molding, etc.).The hardenable fluid can be essentially any fluid that can be induced tosolidify, or that spontaneously solidifies, into a solid capable ofcontaining and/or transporting fluids contemplated for use in and withthe fluidic network. In one embodiment, the hardenable fluid comprises apolymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).Suitable polymeric liquids can include, for example, thermoplasticpolymers, thermoset polymers, waxes, metals, or mixtures or compositesthereof heated above their melting point. As another example, a suitablepolymeric liquid may include a solution of one or more polymers in asuitable solvent, which solution forms a solid polymeric material uponremoval of the solvent, for example, by evaporation. Such polymericmaterials, which can be solidified from, for example, a melt state or bysolvent evaporation, are well known to those of ordinary skill in theart. A variety of polymeric materials, many of which are elastomeric,are suitable, and are also suitable for forming molds or mold masters,for embodiments where one or both of the mold masters is composed of anelastomeric material. A non-limiting list of examples of such polymersincludes polymers of the general classes of silicone polymers, epoxypolymers, and acrylate polymers. Epoxy polymers are characterized by thepresence of a three-membered cyclic ether group commonly referred to asan epoxy group, 1,2-epoxide, or oxirane. For example, diglycidyl ethersof bisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are used in certain embodiments, for example, thesilicone elastomer polydimethylsiloxane. Non-limiting examples of PDMSpolymers include those sold under the trademark Sylgard by Dow ChemicalCo., Midland, Mich., and particularly Sylgard 182, Sylgard 184, andSylgard 186. Silicone polymers including PDMS have several beneficialproperties simplifying fabrication of the microfluidic structures of theinvention. For instance, such materials are inexpensive, readilyavailable, and can be solidified from a prepolymeric liquid via curingwith heat. For example, PDMSs are typically curable by exposure of theprepolymeric liquid to temperatures of about, for example, about 65° C.to about 75° C. for exposure times of, for example, about an hour. Also,silicone polymers, such as PDMS, can be elastomeric and thus may beuseful for forming very small features with relatively high aspectratios, necessary in certain embodiments of the invention. Flexible(e.g., elastomeric) molds or masters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be fabricated and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in an article entitled “Rapid Prototyping ofMicrofluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480,1998 (Duffy et al.), incorporated herein by reference.

Another advantage to forming microfluidic structures of the invention(or interior, fluid-contacting surfaces) from oxidized silicone polymersis that these surfaces can be much more hydrophilic than the surfaces oftypical elastomeric polymers (where a hydrophilic interior surface isdesired). Such hydrophilic channel surfaces can thus be more easilyfilled and wetted with aqueous solutions than can structures comprisedof typical, unoxidized elastomeric polymers or other hydrophobicmaterials.

In some embodiments, the device may be an electrical and/or a mechanicaldevice applicable or affixable to the surface of the skin, e.g., usingadhesive, or other techniques such as those described herein. As anotherexample, the device may be a handheld device that is applied to thesurface of the skin of a subject. In some cases, however, the device maybe sufficiently small or portable that the subject can self-administerthe device. In certain embodiments, the device may also be powered. Insome instances, the device may be applied to the surface of the skin,and is not inserted into the skin. In other embodiments, however, atleast a portion of the device may be inserted into the skin, forexample, mechanically. For example, in one embodiment, the device mayinclude a cutter, such as a hypodermic needle, a knife blade, a piercingelement (e.g., a solid or hollow needle), or the like, as discussedherein.

In some cases, the device may be designed such that portions of thedevice are separable. For example, a first portion of the device may beremoved from the surface of the skin, leaving other portions of thedevice behind on the skin. In one embodiment, a stop may also beincluded to prevent or control the depth to which the cutter or otherdevice inserts into the skin, e.g., to control penetration to theepidermis, dermis, etc.

Accordingly, as described herein, devices of the invention can besingle-stage or multi-stage in some cases. That is, the device candefine a single unit that includes one or more components integrallyconnected to each other which cannot readily be removed from each otherby a user, or can include one or more components which are designed tobe and can readily be removed from each other. As a non-limiting exampleof the later, a two-stage patch can be provided for application to theskin of a subject. The patch can include a first stage designed toreside proximate the skin of the subject for the duration of theanalysis, which might include an analysis region, a reservoir or othermaterial for creating vacuum or otherwise promoting the flow of fluid orother materials relative to the analysis region, a needle or amicroneedle to access interstitial fluid via suction blister or withouta suction blister or the like. A second stage or portion of the devicecan be provided that can initiate operation of the device. For example,the two stage device can be applied to the skin of the user. A button orother component or switch associated with the second portion of thedevice can be activated by the subject to cause insertion of a needle ora microneedle to the skin of the subject, or the like. Then, the secondstage can be removed, e.g., by the subject, and the first stage canremain on the skin to facilitate analysis. In another arrangement, atwo-stage device can be provided where the first stage includesvisualization or other signal-producing components and the second stageincludes components necessary to facilitate the analysis, e.g., thesecond stage can include all components necessary to access bodilyfluid, transport the fluid (if necessary) to a site of analysis, and thelike, and that stage can be removed, leaving only a visualization stagefor the subject or another entity to view or otherwise analyze asdescribed herein.

Any or all of the arrangements described herein can be providedproximate a subject, for example on or proximate a subject's skin.Activation of the devices can be carried out as described herein. Forexample, an on-skin device can be in the form of a patch or the like,optionally including multiple layers for activation, sensing, fluidflow, etc. Activation of the devices can be carried out in a variety ofways. In one manner, a patch can be applied to a subject and a region ofthe patch activated (e.g., tapped by a user) to inject a needle or amicroneedle so as to access interstitial fluid. The same or a differenttapping or pushing action can activate a vacuum source, open and/orclose one or more of a variety of valves, or the like. The device can bea simple one in which it is applied to the skin and operatesautomatically (where e.g., application to the skin access interstitialfluid and draws interstitial fluid into an analysis region) or the patchor other device can be applied to the skin and one tapping or otheractivation can cause fluid to flow through administration of a needle ora microneedle, opening of a valve, activation of vacuum, or anycombination. Any number of activation protocols can be carried out by auser repeatedly pushing or tapping a location or selectively,sequentially, and/or periodically activating a variety of switches(e.g., tapping regions of a patch). In another arrangement, activationof needles or microneedles, creation of suction blisters, opening and/orclosing of valves, and other techniques to facilitate one or moreanalysis can be carried out electronically or in other mannersfacilitated by the subject or by an outside controlling entity. Forexample, a device or patch can be provided proximate a subject's skinand a radio frequency, electromagnetic, or other signal can be providedby a nearby controller or a distant source to activate any of theneedles, blister devices, valves or other components of the devicesdescribed so that any assay or assays can be carried out as desired.

As discussed, various devices of the invention include various systemsand methods for delivering and/or withdrawing fluid from the subject,according to certain embodiments. For instance, the device may comprisea hypodermic needle, a vacuum source, a hygroscopic agent, or the like.Non-limiting examples of suitable delivery techniques include, but arenot limited to, injection (e.g., using needles such as hypodermicneedles) or a jet injector, such as those discussed below. For instance,in one embodiment, the fluid is delivered and/or withdrawn manually,e.g., by manipulating a plunger on a syringe. In another embodiment, thefluid can be delivered and/or withdrawn from the skin mechanically orautomatically, e.g., using a piston pump or the like. Fluid may also bewithdrawn using vacuums such as those discussed herein. For example,vacuum may be applied to a conduit, such as a needle, in fluidiccommunication with interstitial fluid, e.g., within a pooled region offluid, in order to draw up at least a portion of the fluid from thepooled region. In yet another embodiment, fluid is withdrawn usingcapillary action (e.g., using a hypodermic needle having a suitablynarrow inner diameter). In still another embodiment, pressure may beapplied to force fluid out of the needle.

In some embodiments, fluids may be delivered to or withdrawn from theskin using vacuum. The vacuum may be an external vacuum source, and/orthe vacuum source may be self-contained within the device. For example,vacuums of at least about 50 mmHg, at least about 100 mmHg, at leastabout 150 mmHg, at least about 200 mmHg, at least about 250 mmHg, atleast about 300 mmHg, at least about 350 mmHg, at least about 400 mmHg,at least about 450 mmHg, at least about 500 mmHg, at least 550 mmHg, atleast 600 mmHg, at least 650 mmHg, at least about 700 mmHg, or at leastabout 750 mmHg may be applied to the skin. As used herein, “vacuum”refers to pressures that are below atmospheric pressure.

In one set of embodiments, a pressure differential (e.g. a vacuum) maybe created by a pressure regulator. As used here, “pressure regulator”is a pressure controller component or system able to create a pressuredifferential between two or more locations. The pressure differentialshould be at least sufficient to urge the movement of fluid or othermaterial in accordance with various embodiments of the invention asdiscussed herein, and the absolute pressures at the two or morelocations are not important so long as their differential isappropriate, and their absolute values are reasonable for the purposesdiscussed herein. For example, the pressure regulator may produce apressure higher than atmospheric pressure in one location, relative to alower pressure at another location (atmospheric pressure or some otherpressure), where the differential between the pressures is sufficient tourge fluid in accordance with the invention. In another example, theregulator or controller will involve a pressure lower than atmosphericpressure (a vacuum) in one location, and a higher pressure at anotherlocation(s) (atmospheric pressure or a different pressure) where thedifferential between the pressures is sufficient to urge fluid inaccordance with the invention. Wherever “vacuum” or “pressure” is usedherein, in association with a pressure regulator or pressuredifferential of the invention, it should be understood that the oppositecan be implemented as well, as would be understood by those of ordinaryskill in the art, i.e., a vacuum chamber can be replaced in manyinstances with a pressure chamber, for creating a pressure differentialsuitable for urging the movement of fluid or other material.

The pressure regulator may be an external source of vacuum (e.g. a lab,clinic, hospital, etc., house vacuum line or external vacuum pump), amechanical device, a vacuum chamber, pre-packaged vacuum chamber, or thelike. Vacuum chambers can be used in some embodiments, where the devicecontains, e.g., regions in which a vacuum exits or can be created (e.g.a variable volume chamber, a change in volume of which will affectvacuum or pressure). A vacuum chamber can include pre-evacuated (i.e.,pre-packaged) chambers or regions, and/or self-contained actuators.

A “self-contained” vacuum (or pressure) regulator means one that isassociated with (e.g., on or within) the device, e.g. one that definesan integral part of the device, or is a separate component constructedand arranged to be specifically connectable to the particular device toform a pressure differential (i.e., not a connection to an externalsource of vacuum such as a hospital's, clinic's, or lab's house vacuumline, or a vacuum pump suitable for very general use). In someembodiments, the self-contained vacuum source may be actuated in somefashion to create a vacuum within the device. For instance, theself-contained vacuum source may include a piston, a syringe, amechanical device such as a vacuum pump able to create a vacuum withinthe device, and/or chemicals or other reactants that can react toincrease or decrease pressure which, with the assistance of mechanicalor other means driven by the reaction, can form a pressure differentialassociated with a pressure regulator. Chemical reaction can also drivemechanical actuation with or without a change in pressure based on thechemical reaction itself. A self-contained vacuum source can alsoinclude an expandable foam, a shape memory material, or the like.

One category of self-contained vacuum or pressure regulators of theinvention includes self-contained assisted regulators. These areregulators that, upon actuation (e.g., the push of a button, orautomatic actuation upon, e.g., removal from a package or urging adevice against the skin), a vacuum or pressure associated with thedevice is formed where the force that pressurizes or evacuates a chamberis not the same as the actuation force. Examples of self-containedassisted regulators include chambers evacuated by expansion driven by aspring triggered by actuation, release of a shape-memory material orexpandable material upon actuation, initiation of a chemical reactionupon actuation, or the like.

Another category of self-contained vacuum or pressure regulators of theinvention are devices that are not necessarily pre-packaged withpressure or vacuum, but which can be pressurized or evacuated, e.g. by asubject, health care professional at a hospital or clinic prior to use,e.g. by connecting a chamber of the device to a source of vacuum orpressure. For example, the subject, or another person, may actuate thedevice to create a pressure or vacuum within the device, for example,immediately prior to use of the device.

The vacuum or pressure regulator may be a “pre-packaged” pressure orvacuum chamber in the device when used (i.e., the device can be providedready for use by a subject or practitioner with an evacuated region onor in the device, without the need for any actuation to form the initialvacuum). A pre-packaged pressure or vacuum chamber regulator can, e.g.,be a region evacuated (relative to atmospheric pressure) uponmanufacture and/or at some point prior to the point at which it is usedby a subject or practitioner. For example, a chamber is evacuated uponmanufacture, or after manufacture but before delivery of the device tothe user, e.g. the clinician or subject. For instance, in someembodiments, the device contains a vacuum chamber having a vacuum of atleast about 50 mmHg, at least about 100 mmHg, at least about 150 mmHg,at least about 200 mmHg, at least about 250 mmHg, at least about 300mmHg, at least about 350 mmHg, at least about 400 mmHg, at least about450 mmHg, at least about 500 mmHg, at least about 550 mmHg, at leastabout 600 mmHg, at least about 650 mmHg, at least about 700 mmHg, or atleast about 750 mmHg below atmospheric pressure.

In some cases, the device may be applicable or affixable to the surfaceof the skin. For example, in one set of embodiments, the device mayinclude a support structure that contains an adhesive that can be usedto immobilize the device to the skin. The adhesive may be permanent ortemporary, and may be used to affix the device to the surface of theskin. The adhesive may be any suitable adhesive, for example, a pressuresensitive adhesive, a contact adhesive, a permanent adhesive, a hydrogeladhesive, a cyanoacrylate, glue, gum, hot melts, epoxy, or the like. Inmost cases, the adhesive is chosen to be biocompatible orhypoallergenic.

In another set of embodiments, the device may be mechanically held tothe skin, for example, the device may include mechanical elements suchas straps, belts, buckles, strings, ties, elastic bands, or the like.For example, a strap may be worn around the device to hold the device inplace against the skin of the subject. In yet another set ofembodiments, a combination of these and/or other techniques may be used.As one non-limiting example, the device may be affixed to a subject'sarm or leg using adhesive and a strap.

In some embodiments, the device may include a support structure forapplication to the skin of the subject. The support structure may beused, as discussed herein, for applying the fluid transporter to thesurface of the skin of the subject, e.g., so that fluid may be deliveredand/or withdrawn from the skin of the subject. In some cases, thesupport structure may immobilize the fluid transporter such that thefluid transporter cannot move relative to the support structure; inother cases, however, the fluid transporter may be able to move relativeto the support structure. In one embodiment, as a non-limiting example,the fluid transporter is immobilized relative to the support structure,and the support structure is positioned within the device such thatapplication of the device to the skin causes at least a portion of thefluid transporter to pierce the skin of the subject.

For instance, in one set of embodiments, the support structure, or aportion of the support structure, may move from a first position to asecond position. For example, the first position may be one where thesupport structure has been immobilized relative thereto a fluidtransporter does not contact the skin (e.g., the fluid transporter maybe contained within a recess), while the second position may be onewhere the fluid transporter does contact the skin, and in some cases,the fluid transporter may pierce the skin. The support structure may bemoved using any suitable technique, e.g., manually, mechanically,electromagnetically, using a servo mechanism, or the like. In one set ofembodiments, for example, the support structure may be moved from afirst position to a second position by pushing a button on the device,which causes the support structure to move (either directly, or througha mechanism linking the button with the support structure). Othermechanisms (e.g., dials, etc., as discussed herein) may be used inconjunction of or instead of a button. In another set of embodiments,the support structure may be moved from a first position to a secondposition automatically, for example, upon activation by a computer, uponremote activation, after a period of time has elapsed, or the like. Forexample, in one embodiment, a servo connected to the support structureis activated electronically, moving the support structure from the firstposition to the second position.

In some cases, the support structure may also be moved from the secondposition to the first position. For example, after fluid has beendelivered and/or withdrawn from the skin, e.g., using a fluidtransporter the support structure may be moved, which may move the fluidtransporter away from contact with the skin. The support structure maybe moved from the second position to the first position using anysuitable technique, including those described above, and the techniquefor moving the support structure from the second position to the firstposition may be the same or different as that moving the supportstructure from the first position to the second position.

In some cases, the support structure may be able to draw skin towardsthe fluid transporter. For example, in one set of embodiments, thesupport structure may include a vacuum interface, such as is describedherein. The interface may be connected with a vacuum source (externaland/or internal to the device), and when a vacuum is applied, skin maybe drawn towards the support structure, e.g., for contact with a fluidtransporter, such as one or more needles and/or microneedles.

In certain embodiments, the device may also contain an activator. Theactivator may be constructed and arranged to cause exposure of the fluidtransporter to the skin upon activation of the activator. For example,the activator may cause a chemical to be released to contact the skin, aneedle to be driven into the skin, a vacuum to be applied to the skin, ajet of fluid to be directed to the skin, or the like. The activator maybe activated by the subject, and/or by another person (e.g., a healthcare provider), or the device itself may be self-activating, e.g., uponapplication to the skin of a subject. The activator may be activatedonce, or multiple times in some cases.

The device may be activated, for example, by pushing a button, pressinga switch, moving a slider, turning a dial, or the like. The subject,and/or another person, may activate the activator. In some cases, thedevice may be remotely activated. For example, a health care providermay send an electromagnetic signal which is received by the device inorder to activate the device, e.g., a wireless signal, a Bluetoothsignal, an Internet signal, a radio signal, etc.

In one set of embodiments, the device may also include a sensor, forexample embedded within or integrally connected to the device, orpositioned remotely but with physical, electrical, and/or opticalconnection with the device so as to be able to sense a compartmentwithin the device. For example, the sensor may be in fluidiccommunication with fluid withdrawn from a subject, directly, via amicrofluidic channel, an analytical chamber, etc. The sensor may be ableto sense an analyte, e.g., one that is suspected of being in a fluidwithdrawn from a subject. For example, a sensor may be free of anyphysical connection with the device, but may be positioned so as todetect the results of interaction of electromagnetic radiation, such asinfrared, ultraviolet, or visible light, which has been directed towarda portion of the device, e.g., a compartment within the device. Asanother example, a sensor may be positioned on or within the device, andmay sense activity in a compartment by being connected optically to thecompartment. Sensing communication can also be provided where thecompartment is in communication with a sensor fluidly, optically orvisually, thermally, pneumatically, electronically, or the like, so asto be able to sense a condition of the compartment. As one example, thesensor may be positioned downstream of a compartment, within a channelsuch a microfluidic channel, or the like.

The sensor may be, for example, a pH sensor, an optical sensor, anoxygen sensor, a sensor able to detect the concentration of a substance,or the like. Other examples of analytes that the sensor may be used todetermine include, but are not limited to, metal ions, proteins, nucleicacids (e.g. DNA, RNA, etc.), drugs, sugars (e.g., glucose), hormones(e.g., estradiol, estrone, progesterone, progestin, testosterone,androstenedione, etc.), carbohydrates, or other analytes of interest.Non-limiting examples of sensors useful in the invention includedye-based detection systems, affinity-based detection systems,microfabricated gravimetric analyzers, CCD cameras, optical detectors,optical microscopy systems, electrical systems, thermocouples andthermistors, pressure sensors, etc. Those of ordinary skill in the artwill be able to identify other sensors for use in the invention. Thesensor can include a colorimetric detection system in some cases, whichmay be external to the device, or microfabricated into the device incertain cases. As an example of a colorimetric detection system, if adye or a fluorescent entity is used (e.g. in a particle), thecolorimetric detection system may be able to detect a change or shift inthe frequency and/or intensity of the dye or fluorescent entity.

As described herein, any of a variety of signaling or display methods,associated with analyses, can be provided including signaling visually,by smell, sound, feel, taste, or the like, in one set of embodiments.Signal structures and generators include, but are not limited to,displays (visual, LED, light, etc.), speakers, chemical-releasingcompartments (e.g., containing a volatile chemical), mechanical devices,heaters, coolers, or the like. In some cases, the signal structure orgenerator may be integral with the device (e.g., integrally connectedwith a support structure for application to the skin of the subject,e.g., containing a fluid transporter such as a needle or a microneedle),or the signal structure or generator may not be integrally connectedwith the support structure. As used herein, a “signal structure” or a“signal generator” is any apparatus able to generate a signal that isrelated to a condition of a medium. For example, the medium may be abodily fluid, such as blood or interstitial fluid.

In some embodiments, signaling methods such as these may be used toindicate the presence and/or concentration of an analyte determined bythe sensor, e.g., to the subject, and/or to another entity, such asthose described below. Where a visual signal is provided, it can beprovided in the form of change in opaqueness, a change in intensity ofcolor and/or opaqueness, or can be in the form of a message (e.g.,numerical signal, or the like), an icon (e.g., signaling by shape orotherwise a particular medical condition), a brand, logo, or the like.For instance, in one embodiment, the device may include a display. Awritten message such as “take next dose,” or “glucose level is high” ora numerical value might be provided, or a message such as “toxin ispresent.” These messages, icons, logos, or the like can be provided asan electronic read-out by a component of a device and/or can bedisplayed as in inherent arrangement of one or more components of thedevice.

In some embodiments, a device is provided where the device determines aphysical condition of a subject and produces a signal related to thecondition that can be readily understood by the subject (e.g., byprovision of a visual “OK” signal as described above) or can be designedso as not to be readily understandable by a subject. Where not readilyunderstandable, the signal can take a variety of forms. In one form, thesignal might be a series of letters or numbers that mean nothing to thesubject (e.g., A1278CDQ) which would have meaning to a medicalprofessional or the like (and/or be decodable by the same, e.g., withreference to a suitable decoder) and can be associated with a particularphysiological condition. Alternatively, a signal in the form of bar codecan be provided by a device such that, under a particular condition orset of conditions the bar code appears and/or disappears, or changes,and can be read by a bar code reader to communicate information aboutthe subject or analyte. In another embodiment, the device can bedesigned such that an ultraviolet signal is produced, or a signal thatcan be read only under ultraviolet light (e.g., a simple spot or patch,or any other signal such as a series of number, letters, bar code,message, or the like that can be readily understandable or not readilyunderstandable by a subject) can be provided. The signal may beinvisible to the human eye but, upon application UV light or otherexcitation energy, may be readable. The signal can be easily readable orunderstandable by a user via visual observation, or with other sensoryactivity such as smell, feel, etc. In another set of embodimentsequipment as described above may be needed to determine a signalprovided by the device, such as equipment in a clinical setting, etc. Insome cases, the device is able to transmit a signal indicative of theanalyte to a receiver, e.g., as a wireless signal, a Bluetooth signal,an Internet signal, a radio signal, etc.

In some embodiments, quantitative and/or qualitative analyses can beprovided by a device. That is, the device in some cases may provideanalyses that allow “yes/no” tests or the like, or tests that provideinformation on the quantity, concentration, or level of a particularanalyte or analytes. Display configurations can be provided by theinvention that reflect the amount of a particular analyte present in asubject at a particular point in time, or any other variable (presenceof analysis over time, type of analyte, etc.) display configurations cantake a variety of forms. In one example, a dial can be provided, similarto that of a speedometer with a series of level indications (e.g.,numbers around the dial) and a “needle” or other device that indicates aparticular level. In other configurations, a particular area of thedevice (e.g., on a display) can exist that is filled in to a greater orlesser extent depending upon the presence and/or quantity of aparticular analyte present, e.g., in the form of a bar graph. In anotherarrangement a “color wheel” can be provided where the amount of aparticular analyte present can control which colors of the wheel arevisible. Or, different analytes can cause different colors of a wheel ordifferent bars of a graph to become visible or invisible in a multipleanalyte analysis. Multiple-analyte quantitative analyses can bereflected in multiple color wheels, a single color wheel with differentcolors per analyte where the intensity of each color reflects the amountof the analyte, or, for example, a plurality of bar graphs where eachbar graph is reflective of a particular analyte and the level of the bar(and/or degree to which an area is filled in with visible color or othervisible feature) is reflective of the amount of the analyte. As with allembodiments here, whatever signal is displayed can be understandable ornot understandable to any number of participants. For example, it can beunderstandable to a subject or not understandable to a subject. Wherenot understandable it might need to be decoded, read electronically, orthe like. Where read electronically, for example, a device may provide asignal that is not understandable to a subject or not even visible orotherwise able to be sensed by a subject, and a reader can be providedadjacent or approximate to the device that can provide a visible signalthat is understandable or not understandable to the subject, or cantransmit a signal to another entity for analysis.

In connection with any signals associated with any analyses describedherein, another, potentially related signal or other display (or smell,taste, or the like) can be provided which can assist in interpretingand/or evaluating the signal. In one arrangement, a calibration orcontrol is provided proximate to (or otherwise easily comparable with) asignal, e.g., a visual calibration/control or comparator next to orclose to a visual signal provided by a device and/or implanted agents,particles, or the like.

A visual control or reference can be used with another sensory signal,such as that of smell, taste, temperature, itch, etc. Areference/control and/or experimental confirmation component can beprovided, to be used in connection with an in-skin test or vice versa.References/indicators can also be used to indicate the state of life ofa device, changing color or intensity and/or changing in anothersignaling aspect as the device changes relative to its useful life, sothat a user can determine when the device should no longer be reliedupon and/or removed. For certain devices, an indicator or control can beeffected by adding analyte to the control (e.g., from a source outsideof the source to be determine) to confirm operability of the deviceand/or to provide a reference against which to measure a signal of thedevice. For example, a device can include a button to be tapped by auser which will allow an analyte from a reservoir to transfer to anindicator region to provide a signal, to demonstrate operability of thedevice and/or provide a comparator for analysis.

Many of the embodiments described herein involve a quantitative analysisand related signal, i.e., the ability to determine the relative amountor concentration of an analyte in a medium. This can be accomplished ina variety of ways. For example, where an agent (e.g. a binding partnerattached to a nanoparticle) is used to capture and analyze an analyte,the agent can be provided in a gradient in concentration across asensing region of the device. Or a sensing region can include a membraneor other apparatus through which analyte is required to flow or passprior to capture and identification, and the pathway for analyte travelcan vary as a function of position of display region. For example, amembrane can be provided across a sensing region, through which analytemust pass prior to interacting with a layer of binding and/or signalingagent, and the membrane may vary in thickness laterally in a directionrelated to “bar graph” readout. Where a small amount of analyte ispresent, it may pass through the thinner portion but not the thickerportion of the membrane, but where a larger amount is present, it maypass across a thicker portion. The boundary (where one exists) between aregion through which analyte passes, and one through which it does notcompletely pass, can define the “line” of the bar graph. Other ways ofachieving the same or a similar result can include varying theconcentration of a scavenger or transporter of the analyte, or anintermediate reactive species (between analyte and signaling event),across a membrane or other article, gradient in porosity or selectivityof the membrane, ability to absorb or transport sample fluid, or thelike. These principles, in combination with other disclosure herein, canbe used to facilitate any or all of the quantitative analyses describedherein.

In one set of embodiments, a subject having a condition such as aphysiological condition to be analyzed (or other user, such as medicalpersonnel) reads and/or otherwise determines a signal from a device. Forexample, the device may transmit a signal indicative of a condition ofthe subject and/or the device. Alternatively, or in addition, a signalproduced by a device can be acquired in the form of a representation(e.g. a digitized signal, or the like) and transmitted to another entityfor analysis and/or action. For example, a signal can be produced by adevice, e.g., based on a sensor reading of an analyte, based on fluiddelivered and/or withdrawn from the skin, based on a condition of thedevice, or the like. The signal may represent any suitable data orimage. For example, the signal may represent the presence and/orconcentration of an analyte in fluid withdrawn from a subject, theamount of fluid withdrawn from a subject and/or delivered to thesubject, the number of times the device has been used, the battery lifeof the device, the amount of vacuum left in the device, the cleanlinessor sterility of the device, the identity of the device (e.g., wheremultiple devices are given unique identification numbers, to preventcounterfeiting, accidental exchange of equipment to incorrect users,etc.), or the like. For instance, in one set of embodiments, an image ofthe signal (e.g., a visual image or photograph) can be obtained andtransmitted to a different entity (for example, a user can take a cellphone picture of a signal generated by the device and send it, via cellphone, the other entity).

The other entity that the signal is transmitted to can be a human (e.g.,a clinician) or a machine. In some cases, the other entity may be ableto analyze the signal and take appropriate action. In one arrangement,the other entity is a machine or processor that analyzes the signal andoptionally sends a signal back to the device to give direction as toactivity (e.g., a cell phone can be used to transmit an image of asignal to a processor which, under one set of conditions, transmits asignal back to the same cell phone giving direction to the user, ortakes other action). Other actions can include automatic stimulation ofthe device or a related device to dispense a medicament orpharmaceutical, or the like. The signal to direct dispensing of apharmaceutical can take place via the same used to transmit the signalto the entity (e.g., cell phone) or a different vehicle or pathway.Telephone transmission lines, wireless networks, Internet communication,and the like can also facilitate communication of this type.

As one specific example, a device may be a glucose monitor. As signalmay be generated by the device and an image of the signal captured by acell phone camera and then transmitted via cell phone to a clinician.The clinician may then determine that the glucose (or e.g., insulin)level is appropriate or inappropriate and send a message indicating thisback to the subject via cell phone.

Information regarding the analysis can also be transmitted to the sameor a different entity, or a different location simply by removing thedevice or a portion of the device from the subject and transferring itto a different location. For example, a device can be used in connectionwith a subject to analyze presence and/or amount of a particularanalyte. At some point after the onset of use, the device, or a portionof the device carrying a signal or signals indicative of the analysis oranalyses, can be removed and, e.g., attached to a record associated withthe subject. As a specific example, a patch or other device can be wornby a subject to determine presence and/or amount of one or more analytesqualitatively, quantitatively, and/or over time. The subject can visit aclinician who can remove the patch (or other device) or a portion of thepatch and in some cases, attach it to a medical record associated withthe subject.

According to various sets of embodiments, the device may be used one, ormultiple times, depending on the application. For instance, obtainingsamples for sensing, according to certain embodiments of the invention,can be done such that sensing can be carried out continuously,discretely, or a combination of these. For example, where a bodily fluidsuch as blood or interstitial fluid is accessed for determination of ananalyte, fluid can be accessed discretely (i.e., as a single dose, onceor multiple times), or continuously by creating a continuous flow offluid which can be analyzed once or any number of times. Additionally,testing can be carried out once, at a single point in time, or atmultiple points in time, and/or from multiple samples (e.g., at multiplelocations relative to the subject).

Alternatively or in addition, testing can be carried out continuouslyover any number of points in time involving one or any number oflocations relative to the subject or other multiple samples. As anexample, one bolus or isolated sample, of fluid such as interstitialfluid can be obtained. From that fluid a test can be carried out todetermine whether a particular analyte or other agent exists in thefluid. Alternatively, two or more tests can be carried out involvingthat quantity of fluid to determine the presence and/or quantity of twoor more analytes, and any number of such tests can be carried out. Testsinvolving that quantity of fluid can be carried out simultaneously orover a period of time. For example, a test for a particular analyte canbe carried out at various points in time to determine whether the resultchanges over time, or different analytes can be determined at differentpoints in time. As another example, a pool of fluid can be formedbetween layers of skin via, e.g., a suction blister and either withinthe suction blister or from fluid drawn from the suction blister andplaced elsewhere, any of the above and other analysis can be carried outat one or more points in time. Where a suction blister is formed in sucha way that interstitial fluid within the blister changes over time(where an equilibrium exists between interstitial fluid within thesubject and interstitial fluid in the suction blister itself, i.e., thefluid within the blister is ever changing to reflect the content of theinterstitial fluid of the subject in the region of the blister overtime). Testing of fluid within or from the suction blister at variouspoints in time can provide useful information.

In another example, a needle or a microneedle, or other device(s) can beused to access a fluid of a subject such as interstitial fluid (with orwithout use of a suction blister). Fluid can be drawn to a point ofanalysis and analyzed in any manner described herein. For example, ananalysis can be carried out once, to determine the presence and/orquantity of a single analyte, or a number of tests can be carried out.From a single sample of fluid, a particular test or number of tests canbe carried out essentially simultaneously, or analyses can be carriedout over time. Moreover, fluid can be drawn continuously from thesubject and one or more tests can be carried out of any number of pointsin time. A variety of reasons for carrying out one or more tests overthe course of time exists, as would be understood by those of ordinaryskill in the art. One such reason is to determine whether the quantityor another characteristic of an analyte is constant in a subject, orchanges over time. A variety of specific techniques for continuousand/or discrete testing will be described herein.

In some cases, the device may comprise a cutter able to cut or piercethe surface of the skin. The cutter may comprise any mechanism able tocreate a path to a fluid within the skin, e.g., through which fluids maybe delivered and/or removed from the skin. For example, the cutter maycomprise a hypodermic needle, a knife blade, a piercing element (e.g., asolid or a hollow needle), or the like, which can be applied to the skinto create a suitable conduit for the withdrawal of fluid from the skin.In one embodiment, a cutter is used to create such a pathway andremoved, then fluid is removed via this pathway using any suitabletechnique. In another embodiment, the cutter remains in place within theskin, and fluid may be drawn through a conduit within the cutter.

In some embodiments, fluid may be withdrawn using an electric charge.For example, reverse iontophoresis may be used. Without wishing to bebound by any theory, reverse iontophoresis uses a small electric currentto drive charged and highly polar compounds across the skin. Since theskin is negatively charged at physiologic pH, it acts as a permselectivemembrane to cations, and the passage of counterions across the skininduces an electroosmotic solvent flow that may carry neutral moleculesin the anode-to-cathode direction. Components in the solvent flow may beanalyzed as described elsewhere herein. In some instances, a reverseiontophoresis apparatus may comprise an anode cell and a cathode cell,each in contact with the skin. The anode cell may be filled, forexample, with an aqueous buffer solution (i.e., aqueous Tris buffer)having a pH greater than 4 and an electrolyte (i.e. sodium chloride).The cathode cell can be filled with aqueous buffer. As one example, afirst electrode (e.g., an anode) can be inserted into the anode cell anda second electrode (e.g., a cathode) can be inserted in the cathodecell. In some embodiments, the electrodes are not in direct contact withthe skin.

A current may be applied to induce reverse iontophoresis, therebyextracting a fluid from the skin. The current applied may be, forexample, greater than 0.01 mA, greater than 0.3 mA, greater than 0.1 mA,greater than 0.3 mA, greater than 0.5 mA, or greater than 1 mA. Itshould be understood that currents outside these ranges may be used aswell. The current may be applied for a set period of time. For example,the current may be applied for greater than 30 seconds, greater than 1minute, greater than 5 minutes, greater than 30 minutes, greater than 1hour, greater than 2 hours, or greater than 5 hours. It should beunderstood that times outside these ranges may be used as well.

In one set of embodiments, the device may comprise an apparatus forablating the skin. Without wishing to be bound by any theory, it isbelieved that ablation comprises removing a microscopic patch of stratumcorneum (i.e., ablation forms a micropore), thus allowing access tobodily fluids. In some cases, thermal, radiofrequency, and/or laserenergy may be used for ablation. In some instances, thermal ablation maybe applied using a heating element. Radiofrequency ablation may becarried out using a frequency and energy capable of heating water and/ortissue. A laser may also be used to irradiate a location on the skin toremove a portion. In some embodiments, the heat may be applied in pulsessuch that a steep temperature gradient exists essentially perpendicularto the surface of the skin. For example, a temperature of at least 100°C., at least 200° C., at least 300° C., or at least 400° C. may beapplied for less than 1 second, less than 0.1 seconds, less than 0.01seconds, less than 0.005 seconds, or less than 0.001 seconds.

In some embodiments, the device may comprise a mechanism for taking asolid sample of tissue. For example, a solid tissue sample may beacquired by methods such as scraping the skin or cutting out a portion.Scraping may comprise a reciprocating action whereby an instrument isscraped along the surface of the skin in two or more directions.Scraping can also be accomplished by a rotating action, for exampleparallel to the surface of the skin and in one direction (i.e., with aroller drum) or parallel to the surface of the skin and in a circularmanner (i.e., with a drilling instrument). A cutting mechanism maycomprise a blade capable of making one or more incisions and a mechanismfor removing a portion of tissue (i.e., by suction or mechanicallypicking up) or may use a pincer mechanism for cutting out a portion oftissue. A cutting mechanism may also function by a coring action. Forexample, a hollow cylindrical device can be penetrated into the skinsuch that a cylindrical core of tissue may be removed. A solid samplemay be analyzed directly or may be liquefied prior to analysis.Liquefaction can comprise treatment with organic solvents, enzymaticsolutions, etc.

In some cases, the device may contain a shape memory polymer and/ormetal, for example, one that is sensitive to heat. Upon insertion intothe skin between the epidermis and dermis, the shape memory polymer mayexpand in some fashion, allowing separation of the epidermis and thedermis to occur. Non-limiting examples of shape-memory polymers andmetals include Nitinol, compositions of oligo(epsilon-caprolactone)dioland crystallisable oligo(rho-dioxanone)diol, or compositions ofoligo(epsilon-caprolactone)dimethacrylate and n-butyl acrylate. Forexample, the shape memory polymer (or metal) may have a first, condensedshape at a temperature below the body temperature of the subject, butupon insertion into the skin, the cutter heats and assumes a second,larger shape that causes at least some separation to occur between theepidermis and dermis.

The device, in certain embodiments, may also contain a portion able todetermine the fluid removed from the skin. For example, a portion of thedevice may contain a sensor, or reagents able to interact with ananalyte contained or suspected to be present within the withdrawn fluidfrom the subject, for example, a marker for a disease state. Asnon-limiting examples, the sensor may contain an antibody able tointeract with a marker for a disease state, an enzyme such as glucoseoxidase or glucose 1-dehydrogenase able to detect glucose, or the like.The analyte may be determined quantitatively or qualitatively, and/orthe presence or absence of the analyte within the withdrawn fluid may bedetermined in some cases. Those of ordinary skill in the art will beaware of many suitable commercially-available sensors, and the specificsensor used may depend on the particular analyte being sensed. Forinstance, various non-limiting examples of sensor techniques includepressure or temperature measurements, spectroscopy such as infrared,absorption, fluorescence, UV/visible, FTIR (“Fourier Transform InfraredSpectroscopy”), or Raman; piezoelectric measurements; immunoassays;electrical measurements, electrochemical measurements (e.g.,ion-specific electrodes); magnetic measurements, optical measurementssuch as optical density measurements; circular dichroism; lightscattering measurements such as quasielectric light scattering;polarimetry; refractometry; chemical indicators such as dyes; orturbidity measurements, including nephelometry.

As mentioned, certain aspects of the present invention are generallydirected to particles such as anisotropic particles or colloids, whichcan be used in a wide variety of applications. For instance, theparticles may be present within the skin, or externally of the skin,e.g., in a device on the surface of the skin. The particles may includemicroparticles and/or nanoparticles. As discussed above, a“microparticle” is a particle having an average diameter on the order ofmicrometers (i.e., between about 1 micrometer and about 1 mm), while a“nanoparticle” is a particle having an average diameter on the order ofnanometers (i.e., between about 1 nm and about 1 micrometer. Theparticles may be spherical or non-spherical, in some cases. For example,the particles may be oblong or elongated, or have other shapes such asthose disclosed in U.S. patent application Ser. No. 11/851,974, filedSep. 7, 2007, entitled “Engineering Shape of Polymeric Micro- andNanoparticles,” by S. Mitragotri, et al.; International PatentApplication No. PCT/US2007/077889, filed Sep. 7, 2007, entitled“Engineering Shape of Polymeric Micro- and Nanoparticles,” by S.Mitragotri, et al., published as WO 2008/031035 on Mar. 13, 2008; U.S.patent application Ser. No. 11/272,194, filed Nov. 10, 2005, entitled“Multi-phasic Nanoparticles,” by J. Lahann, et al., published as U.S.Patent Application Publication No. 2006/0201390 on Sep. 14, 2006; orU.S. patent application Ser. No. 11/763,842, filed Jun. 15, 2007,entitled “Multi-Phasic Bioadhesive Nan-Objects as Biofunctional Elementsin Drug Delivery Systems,” by J. Lahann, published as U.S. PatentApplication Publication No. 2007/0237800 on Oct. 11, 2007, each of whichis incorporated herein by reference.

An “anisotropic” particle, as used herein, is one that is notspherically symmetric (although the particle may still exhibit varioussymmetries), although the particle may have sufficient asymmetry tocarry out at least some of the goals of the invention as describedherein. On the basis of the present disclosure, this will be clearlyunderstood by those of ordinary skill in the art. The asymmetry can beasymmetry of shape, of composition, or both. As an example, a particlehaving the shape of an egg or an American football is not perfectlyspherical, and thus exhibits anisotropy. As another example, a spherepainted such that exactly one half is red and one half is blue (orotherwise presents different surface characteristics on different sides)is also anisotropic, as it is not perfectly spherically symmetric,although it would still exhibit at least one axis of symmetry.

Accordingly, a particle may be anisotropic due to its shape and/or dueto two or more regions that are present on the surface of and/or withinthe particle. For instance, the particle may include a first surfaceregion and a second surface region that is distinct from the firstregion in some way, e.g., due to coloration, surface coating, thepresence of one or more reaction entities, etc. The particle may includedifferent regions only on its surface or the particle may internallyinclude two or more different regions, portions of which extend to thesurface of the particle. The regions may have the same or differentshapes, and be distributed in any pattern on the surface of theparticle. For instance, the regions may divide the particle into twohemispheres, such that each hemisphere has the same shape and/or thesame surface area, or the regions may be distributed in more complexarrangements.

Non-limiting examples of particles can be seen in U.S. patentapplication Ser. No. 11/272,194, filed Nov. 10, 2005, entitled“Multi-phasic Nanoparticles,” by J. Lahann, et al., published as U.S.Patent Application Publication No. 2006/0201390 on Sep. 14, 2006; U.S.patent application Ser. No. 11/763,842, filed Jun. 15, 2007, entitled“Multi-Phasic Bioadhesive Nan-Objects as Biofunctional Elements in DrugDelivery Systems,” by J. Lahann, published as U.S. Patent ApplicationPublication No. 2007/0237800 on Oct. 11, 2007; or U.S. ProvisionalPatent Application Ser. No. 61/058,796, filed Jun. 4, 2008, entitled“Compositions and Methods for Diagnostics, Therapies, and OtherApplications,” by D. Levinson, each of which is incorporated herein byreference.

The particles (which may be anisotropic, or not anisotropic) may beformed of any suitable material, depending on the application. Forexample, the particles may comprise a glass, and/or a polymer such aspolyethylene, polystyrene, silicone, polyfluoroethylene, polyacrylicacid, a polyamide (e.g., nylon), polycarbonate, polysulfone,polyurethane, polybutadiene, polybutylene, polyethersulfone,polyetherimide, polyphenylene oxide, polymethylpentene,polyvinylchloride, polyvinylidene chloride, polyphthalamide,polyphenylene sulfide, polyester, polyetheretherketone, polyimide,polymethylmethacylate and/or polypropylene. In some cases, the particlesmay comprise a ceramic such as tricalcium phosphate, hydroxyapatite,fluorapatite, aluminum oxide, or zirconium oxide. In some cases (forexample, in certain biological applications), the particles may beformed from biocompatible and/or biodegradable polymers such aspolylactic and/or polyglycolic acids, polyanhydride, polycaprolactone,polyethylene oxide, polyacrylamide, polyacrylic acid, polybutyleneterephthalate, starch, cellulose, chitosan, and/or combinations ofthese. In one set of embodiments, the particles may comprise a hydrogel,such as agarose, collagen, or fibrin. The particles may include amagnetically susceptible material in some cases, e.g., a materialdisplaying paramagnetism or ferromagnetism. For instance, the particlesmay include iron, iron oxide, magnetite, hematite, or some othercompound containing iron, or the like. In another embodiment, theparticles can include a conductive material (e.g., a metal such astitanium, copper, platinum, silver, gold, tantalum, palladium, rhodium,etc.), or a semiconductive material (e.g., silicon, germanium, CdSe,CdS, etc.). Other particles potentially useful in the practice of theinvention include ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe,CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, or GaAs. The particles mayinclude other species as well, such as cells, biochemical species suchas nucleic acids (e.g., RNA, DNA, PNA, etc.), proteins, peptides,enzymes, nanoparticles, quantum dots, fragrances, indicators, dyes,fluorescent species, chemicals, small molecules (e.g., having amolecular weight of less than about 1 kDa), or the like.

As an example, certain particles or colloids such as gold nanoparticlescan be coated with agents capable of interacting with an analyte. Suchparticles may associate with each other, or conversely, dissociate inthe presence of analyte in such a manner that a change is conferred uponthe light absorption property of the material containing the particles.This approach can also be used as a skin-based visual sensor, in oneembodiment. A non-limiting example of a technique for identifyingaggregates is disclosed in U.S. patent application Ser. No. 09/344,667,filed Jun. 25, 1999, entitled “Nanoparticles Having OligonucleotidesAttached Thereto and Uses Therefor,” by Mirkin, et al., now U.S. Pat.No. 6,361,944, issued Mar. 26, 2002.

The particles may also have any shape or size. For instance, theparticles may have an average diameter of less than about 5 mm or 2 mm,or less than about 1 mm, or less than about 500 microns, less than about200 microns, less than about 100 microns, less than about 60 microns,less than about 50 microns, less than about 40 microns, less than about30 microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.As discussed, the particles may be spherical or non-spherical. Theaverage diameter of a non-spherical particle is the diameter of aperfect sphere having the same volume as the non-spherical particle. Ifthe particle is non-spherical, the particle may have a shape of, forinstance, an ellipsoid, a cube, a fiber, a tube, a rod, or an irregularshape. In some cases, the particles may be hollow or porous. Othershapes are also possible, for instance, core/shell structures (e.g.,having different compositions), rectangular disks, high aspect ratiorectangular disks, high aspect ratio rods, worms, oblate ellipses,prolate ellipses, elliptical disks, UFOs, circular disks, barrels,bullets, pills, pulleys, biconvex lenses, ribbons, ravioli, flat pills,bicones, diamond disks, emarginate disks, elongated hexagonal disks,tacos, wrinkled prolate ellipsoids, wrinkled oblate ellipsoids, porousellipsoid disks, and the like. See, e.g., International PatentApplication No. PCT/US2007/077889, filed Sep. 7, 2007, entitled“Engineering Shape of Polymeric Micro- and Nanoparticles,” by S.Mitragotri, et al., published as WO 2008/031035 on Mar. 13, 2008,incorporated herein by reference.

In one aspect of the invention, a particle may include one or morereaction entities present on the surface (or at least a portion of thesurface) of the particle. The reaction entity may be any entity able tointeract with and/or associate with an analyte, or another reactionentity. For instance, the reaction entity may be a binding partner ableto bind an analyte. For example, the reaction entity may be a moleculethat can undergo binding with a particular analyte. The reactionentities may be used, for example, to determine pH or metal ions,proteins, nucleic acids (e.g. DNA, RNA, etc.), drugs, sugars (e.g.,glucose), hormones (e.g., estradiol, estrone, progesterone, progestin,testosterone, androstenedione, etc.), carbohydrates, or other analytesof interest.

The term “binding partner” refers to a molecule that can undergo bindingwith a particular molecule, e.g., an analyte. For example, the bindingmay be highly specific and/or non-covalent. Binding partners which formhighly specific, non-covalent, physiochemical interactions with oneanother are defined herein as “complementary.” Biological bindingpartners are examples. For example, Protein A is a binding partner ofthe biological molecule IgG, and vice versa. Other non-limiting examplesinclude nucleic acid-nucleic acid binding, nucleic acid-protein binding,protein-protein binding, enzyme-substrate binding, receptor-ligandbinding, receptor-hormone binding, antibody-antigen binding, etc.Binding partners include specific, semi-specific, and non-specificbinding partners as known to those of ordinary skill in the art. Forexample, Protein A is usually regarded as a “non-specific” orsemi-specific binder. As another example, the particles may contain anenzyme such as glucose oxidase or glucose 1-dehydrogenase, or a lectinsuch as concanavalin A that is able to bind to glucose.

As additional examples, binding partners may include antibody/antigenpairs, ligand/receptor pairs, enzyme/substrate pairs and complementarynucleic acids or aptamers. Examples of suitable epitopes which may beused for antibody/antigen binding pairs include, but are not limited to,HA, FLAG, c-Myc, glutathione-S-transferase, His₆, GFP, DIG, biotin andavidin. Antibodies may be monoclonal or polyclonal. Suitable antibodiesfor use as binding partners include antigen-binding fragments, includingseparate heavy chains, light chains Fab, Fab′, F(ab′)₂, Fabc, and Fv.Antibodies also include bispecific or bifunctional antibodies. Exemplarybinding partners include biotin/avidin, biotin/streptavidin,biotin/neutravidin and glutathione-S-transferase/glutathione.

The term “binding” generally refers to the interaction between acorresponding pair of molecules or surfaces that exhibit mutual affinityor binding capacity, typically due to specific or non-specific bindingor interaction, including, but not limited to, biochemical,physiological, and/or chemical interactions. The binding may be betweenbiological molecules, including proteins, nucleic acids, glycoproteins,carbohydrates, hormones, or the like. Specific non-limiting examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, virus/cell surface receptor, etc. As another example, thebinding agent may be a chelating agent (e.g., ethylenediaminetetraaceticacid) or an ion selective polymer (e.g., a block copolymer such aspoly(carbonate-b-dimethylsiloxane), a crown ether, or the like). Asanother example, the binding partners may be biotin and streptavidin, orthe binding partners may be various antibodies raised against a protein.

The term “specifically binds,” when referring to a binding partner(e.g., protein, nucleic acid, antibody, etc.), refers to a reaction thatis determinative of the presence and/or identity of one or other memberof the binding pair in a mixture of heterogeneous molecules (e.g.,proteins and other biologics). Thus, for example, in the case of areceptor/ligand binding pair, the ligand would specifically and/orpreferentially select its receptor from a complex mixture of molecules,or vice versa. An enzyme would specifically bind to its substrate, anucleic acid would specifically bind to its complement, an antibodywould specifically bind to its antigen, etc. The binding may be by oneor more of a variety of mechanisms including, but not limited to ionicinteractions or electrostatic interactions, covalent interactions,hydrophobic interactions, van der Waals interactions, etc.

Thus, the invention provides, in certain embodiments, particles that areable to bind to an analyte, e.g., via a binding partner to the analyte,and such particles can be used to determine the analyte. Suchdetermination may occur within the skin, and/or externally of thesubject, e.g., within a device on the surface of the skin, depending onthe embodiment. “Determine,” in this context, generally refers to theanalysis of a species, for example, quantitatively or qualitatively,and/or the detection of the presence or absence of the species. Thespecies may be, for example, a bodily fluid and/or an analyte suspectedof being present in the bodily fluid. “Determining” may also refer tothe analysis of an interaction between two or more species, for example,quantitatively or qualitatively, and/or by detecting the presence orabsence of the interaction, e.g. determination of the binding betweentwo species. “Determining” also means detecting or quantifyinginteraction between species. As an example, an analyte may cause adeterminable change in a property of the particles, e.g., a change in achemical property of the particles, a change in the appearance and/oroptical properties of the particles, a change in the temperature of theparticles, a change in an electrical property of the particles, etc. Insome cases, the change may be one that is determinable by a human,unaided by any equipment that may be directly applied to the human. Forinstance, the determinable change may be a change in appearance (e.g.,color), a change in temperature, the production of an odor, etc., whichcan be determined by a human without the use of any equipment (e.g.,using the eyes). Non-limiting examples include temperature changes,chemical reactions or other interactions (e.g., with capsaicin) that canbe sensed, or the like. Examples of capsaicin and capsaicin-likemolecules include, but are not limited to, dihydrocapsaicin,nordihydrocapsaicin, homodihydrocapsaicin, homocapsaicin, or nonivamide.Without wishing to be bound by any theory, it is believed thatinteractions with capsaicin and capsaicin-like molecules can be sensedby a subject, since such molecules may interact with certain nerveendings, which produces a sensation of burning.

In one set of embodiments, more than one particle may be able to bind ananalyte, and/or more than one analyte may bind to a particle. In somecases, such multiple binding properties may result in the clustering ofmore than one particle to an analyte and/or more than one analyte to aparticle. Such clustering can be determined in some fashion, e.g., via achange in an optical property. As an example, an aggregate of particlesmay form in the absence of analyte, but disaggregate (at leastpartially) in the presence of the analyte, e.g., if the analyte and theparticles exhibit competitive or non-competitive inhibition. Suchbinding and/or aggregation may be equilibrium-based in some cases, i.e.,the binding and/or aggregation occurs in equilibrium with unbinding ordisaggregation processes. Thus, when the environment surrounding theparticles is altered in some fashion (e.g., a change in concentration ofan analyte), the equilibrium may shift in response, which can be readilydetermined (e.g., as a change in color). It should be noted that suchequilibrium-based systems may be able to determine such changes inenvironment, in some cases, without the need to apply any energy todetermine the environmental change. In another example, aggregation maycause a change in an electrical or a magnetic property.

As an example, an optical property of the medium containing the clustersmay be altered in some fashion (e.g., exhibiting different lightscattering properties, different opacities, different degrees oftransparency, etc.), which can be correlated with the analyte. In somecases, the color may change in intensity, for example, the clustering ofparticles may bring two or more reactants into close proximity.

Other properties may also be determined besides color. Accordingly, itshould be understood that the use of “color” with respect to particlesas used herein is by way of example only, and other properties may bedetermined instead of or in addition to color. For instance, clusteringof aniostropic particles may cause a change in an electrical or amagnetic property of the particles, which can be determined bydetermining an electrical or a magnetic field. As another example, thefirst region and the second region may have different reactivities(e.g., the first region may be reactive to an enzyme, an antibody,etc.), and aggregation of the particles may cause a net change in thereactivity. As still another example, size may be used to determine theparticles and/or the analyte. For instance, the aggregates may bevisually identifiable, the aggregates may form a precipitant, or thelike. Thus, for example, the particles (which may be anisotropic or notanisotropic) may appear to be a first color when separate, and a secondcolor when aggregation occurs. In some cases, an assay (e.g., anagglutination assay) may be used to determine the aggregation. Inanother set of embodiments, an ordering of the particles may bedetermined. For example, in the absence of an analyte, the particles maybe ordered on the surface of a substrate; while in the presence of ananalyte, the particles may bind to the analyte and become disorderedrelative to the surface. This ordering may be determined, for example,as a change in an optical property of the surface (e.g., index ofrefraction, color, opacity, etc.). As yet other examples, a shape changemay be produced using a shape memory polymer or a “smart polymer,” andthis may be able to be sensed by feel. Alternatively, a color may bereleased, a hydrolysis reaction may occur, or aggregation of theparticles may occur.

In one embodiment, the binding or presence of the analyte, e.g. presentin interstitial fluid optionally created using a suction blister device,results in a tactile change (e.g., change in shape or texture). Forexample, shape memory polymer (SMPs) can be used to detect the presenceof one or more analytes. SMPs are generally characterized as phasesegregated linear block co-polymers having a hard segment and a softsegment. The hard segment is typically crystalline, with a definedmelting point, and the soft segment is typically amorphous, with adefined glass transition temperature. In some embodiments, however, thehard segment is amorphous and has a glass transition temperature ratherthan a melting point. In other embodiments, the soft segment iscrystalline and has a melting point rather than a glass transitiontemperature. The melting point or glass transition temperature of thesoft segment is substantially less than the melting point or glasstransition temperature of the hard segment.

When the SMP is heated above the melting point or glass transitiontemperature of the hard segment, the material can be shaped. This(original) shape can be “memorized” by cooling the SMP below the meltingpoint or glass transition temperature of the hard segment. When theshaped SMP is cooled below the melting point or glass transitiontemperature of the soft segment while the shape is deformed, that(temporary) shape is fixed. The original shape is recovered by heatingthe material above the melting point or glass transition temperature ofthe soft segment but below the melting point or glass transitiontemperature of the hard segment. The recovery of the original shape,which is induced by an increase in temperature, is called the thermalshape memory effect. Properties that describe the shape memorycapabilities of a material include the shape recovery of the originalshape and the shape fixity of the temporary shape

Shape memory polymers can contain at least one physical crosslink(physical interaction of the hard segment) or contain covalentcrosslinks instead of a hard segment. The shape memory polymers also canbe interpenetrating networks or semi-interpenetrating networks. Inaddition to changes in state from a solid to liquid state (melting pointor glass transition temperature), hard and soft segments may undergosolid to solid state transitions, and can undergo ionic interactionsinvolving polyelectrolyte segments or supramolecular effects based onhighly organized hydrogen bonds.

Other polymers that can shape or phase change as a function oftemperature include PLURONICS®. These are also known as poloxamers,nonionic triblock copolymers composed of a central hydrophobic chain ofpolyoxypropylene (poly(propylene oxide)) flanked by two hydrophilicchains of polyoxyethylene (poly(ethylene oxide)). Because the lengths ofthe polymer blocks can be customized, many different poloxamers existthat have slightly different properties. For the generic term“poloxamer,” these copolymers are commonly named with the letter “P”(for poloxamer) followed by three digits, the first two digits×100 givethe approximate molecular mass of the polyoxypropylene core, and thelast digit×10 gives the percentage polyoxyethylene content (e.g.,P407=Poloxamer with a polyoxypropylene molecular mass of 4,000 g/mol anda 70% polyoxyethylene content). For the PLURONICS® tradename, coding ofthese copolymers starts with a letter to define its physical form atroom temperature (L=liquid, P=paste, F=flake (solid)) followed by two orthree digits. The first digit (two digits in a three-digit number) inthe numerical designation, multiplied by 300, indicates the approximatemolecular weight of the hydrophobe; and the last digit×10 gives thepercentage polyoxyethylene content (e.g., L61=Pluronic with apolyoxypropylene molecular mass of 1,800 g/mol and a 10% polyoxyethylenecontent).

Other temperature sensitive polymers that form gels that have a distinctphase change at its lower critical solution temperature (LCST) includingthe cross-linked copolymers comprising hydrophobic monomers, hydrogenbonding monomers, and thermosensitive monomers.

Additional thermal responsive, water soluble polymers including theco-polymerization product of N-isopropyl acrylamide (NIP);1-vinyl-2-pyrrolidinone (VPD); and optionally, acrylic acid (AA), changeshape as a function of temperature. As the proportion of component AAincreases, the Lower Critical Solution Temperature (LCST) decreases andthe COOH reactive groups increase, which impart high reactivity to thecopolymer. By adjusting the proportion of the monomers, a broad range ofLCST can be manipulated from about 20° C. to 80° C.

While the shape memory effect is typically described in the context of athermal effect, the polymers can change their shape in response toapplication of light, changes in ionic concentration and/or pH, electricfield, magnetic field or ultrasound. For example, a SMP can include atleast one hard segment and at least one soft segment, wherein at leasttwo of the segments, e.g., two soft segments, are linked to each othervia a functional group that may be cleavable under application of light,electric field, magnetic field, or ultrasound. The temporary shape maybe fixed by crosslinking the linear polymers. By cleaving those linksthe original shape can be recovered. The stimuli for crosslinking andcleaving these bonds can be the same or different.

In one embodiment, the shape memory polymer composition binds, complexesto, or interacts with an analyte, which can be a chromophore. The hardand/or soft segments can include double bonds that shift from cis totrans isomers when the chromophores absorb light. Light can therefore beused to detect the presence of a chromophore analyte by observingwhether or not the double bond isomerizes.

The shape memory effect can also be induced by changes in ionic strengthor pH. Various functional groups are known to crosslink in the presenceof certain ions or in response to changes in pH. For example, calciumions are known to crosslink amine and alcohol groups, i.e., the aminegroups on alginate can be crosslinked with calcium ions. Also,carboxylate and amine groups become charged species at certain pHs. Whenthese species are charged, they can crosslink with ions of the oppositecharge. The presence of groups, which respond to changes in theconcentration of an ionic species and/or to changes in pH, on the hardand/or soft segments results in reversible linkages between thesesegments. One can fix the shape of an object while crosslinking thesegments. After the shape has been deformed, alteration of the ionicconcentration or pH can result in cleavage of the ionic interactionswhich formed the crosslinks between the segments, thereby relieving thestrain caused by the deformation and thus returning the object to itsoriginal shape. Because ionic bonds are made and broken in this process,it can only be performed once. The bonds, however, can be re-formed byaltering the ionic concentration and/or pH, so the process can berepeated as desired. Thus, in this embodiment, the presence of ananalyte which changes the ionic strength or pH can induce a shape memoryeffect in the polymer confirming the presence of the analyte.

Electric and/or magnetic fields can also be used to induce a shapememory effect. Various moieties, such as chromophores with a largenumber of delocalized electrons, increase in temperature in response topulses of applied electric or magnetic fields as a result of theincreased electron flow caused by the fields. After the materialsincrease in temperature, they can undergo temperature induced shapememory in the same manner as if the materials were heated directly.These compositions are useful in biomedical applications where thedirect application of heat to an implanted material may be difficult,but the application of an applied magnetic or electric field would onlyaffect those molecules with the chromophore, and not heat thesurrounding tissue. For example, the presence of a chromophore analytewith a large number of delocalized electrons can cause an increase intemperature in the microenvironment surrounding the shape memory polymerimplant in response to pulses of applied electric or magnetic fields.This increase in temperature can in turn cause a thermal shape memoryeffect, thus confirming the presence of a particular analyte.

Other types of “smart polymers” may also be used. The combination of thecapabilities of stimuli-responsive components such as polymers andinteractive molecules to form site-specific conjugates are useful in avariety of assays, separations, processing, and other uses. The polymerchain conformation and volume can be manipulated through alteration inpH, temperature, light, or other stimuli. The interactive molecules canbe biomolecules like proteins or peptides, such as antibodies,receptors, or enzymes, polysaccharides or glycoproteins whichspecifically bind to ligands, or nucleic acids such as antisense,ribozymes, and aptamers, or ligands for organic or inorganic moleculesin the environment or manufacturing processes. The stimuli-responsivepolymers are coupled to recognition biomolecules at a specific site sothat the polymer can be manipulated by stimulation to alterligand-biomolecule binding at an adjacent binding site, for example, thebiotin binding site of streptavidin, the antigen-binding site of anantibody or the active, substrate-binding site of an enzyme. Binding maybe completely blocked (i.e., the conjugate acts as an on-off switch) orpartially blocked (i.e., the conjugate acts as a rheostat to partiallyblock binding or to block binding only of larger ligands). Once a ligandis bound, it may also be ejected from the binding site by stimulatingone (or more) conjugated polymers to cause ejection of the ligand andwhatever is attached to it. Alternatively, selective partitioning, phaseseparation or precipitation of the polymer-conjugated biomolecule can beachieved through exposure of the stimulus-responsive component to anappropriate environmental stimulus.

Liquid crystal polymeric materials can also be used to provide a signalfor detection or quantification of analyte. Liquid crystals arematerials that exhibit long-range order in only one or two dimensions,not all three. A distinguishing characteristic of the liquid crystallinestate is the tendency of the molecules, or mesogens, to point along acommon axis, known as the director. This feature is in contrast tomaterials where the molecules are in the liquid or amorphous phase,which have no intrinsic order, and molecules in the solid state, whichare highly ordered and have little translational freedom. Thecharacteristic orientational order of the liquid crystal state fallsbetween the crystalline and liquid phases. These can be pressure ortemperature sensitive, and react by producing a change in color orshape.

In some cases, the particles may contain a diagnostic agent able todetermine an analyte. An example of an analyte within a subject isglucose (e.g., for diabetics); other potentially suitable analytesinclude ions such as sodium, potassium, chloride, calcium, magnesium,and/or bicarbonate (e.g., to determine dehydration); gases such ascarbon dioxide or oxygen; pH; metabolites such as urea, blood ureanitrogen or creatinine; hormones such as estradiol, estrone,progesterone, progestin, testosterone, androstenedione, etc. (e.g., todetermine pregnancy, illicit drug use, or the like); or cholesterol.Still other potentially suitable analytes include various pathogens suchas bacteria or viruses, and/or markers produced by such pathogens. Forexample, a particle may include an antibody directed at a markerproduced by bacteria. In addition, more than one analyte may bedetermined in a subject, e.g., through the use of different particletypes and/or through the use of particles able to determine more thanone analyte, such as those discussed above. For instance, a first set ofparticles may determine a first analyte and a second set of particlesmay determine a second analyte. In some cases, such particles may beused to determine a physical condition of a subject. For instance, theparticles may exhibit a first color indicating a healthy state and asecond color indicating a disease state. In some cases, the appearanceof the particles may be used to determine a degree of health. Forinstance, the particles may exhibit a first color indicating a healthystate, a second color indicating a warning state, and a third colorindicating a dangerous state, or the particles may exhibit a range ofcolors indicating a degree of health of the subject.

Binding partners to these and/or other species are well-known in theart. Non-limiting examples include pH-sensitive entities such as phenolred, bromothymol blue, chlorophenol red, fluorescein, HPTS,5(6)-carboxy-2′,7′-dimethoxyfluorescein SNARF, and phenothalein;entities sensitive to calcium such as Fura-2 and Indo-1; entitiessensitive to chloride such as 6-methoxy-N-(3-sulfopropyl)-quinolinim andlucigenin; entities sensitive to nitric oxide such as4-amino-5-methylamino-2′,7′-difluorofluorescein; entities sensitive todissolved oxygen such as tris(4,4′-diphenyl-2,2′-bipyridine) ruthenium(II) chloride pentahydrate; entities sensitive to dissolved CO₂;entities sensitive to fatty acids, such as BODIPY 530-labeledglycerophosphoethanolamine; entities sensitive to proteins such as4-amino-4′-benzamidostilbene-2-2′-disulfonic acid (sensitive to serumalbumin), X-Gal or NBT/BCIP (sensitive to certain enzymes), Tb³⁺ fromTbCl₃ (sensitive to certain calcium-binding proteins), BODIPY FLphallacidin (sensitive to actin), or BOCILLIN FL (sensitive to certainpenicillin-binding proteins); entities sensitive to concentration ofglucose, lactose or other components, or entities sensitive toproteases, lactates or other metabolic byproducts, entities sensitive toproteins, antibodies, or other cellular products.

In one set of embodiments, at least some of the particles used in thesubject to determine the analyte are anisotropic particles (in othercases, however, the particles are not necessarily anisotropic), and insome cases, substantially all of the particles are anisotropicparticles. In certain cases, at least about 10%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, or atleast about 99% of the particles are anisotropic particles. In oneembodiment, the anisotropic particles may have a first region having afirst color and a second region having a second color distinct from thefirst color, and the particles, upon exposure to the analyte within thesubject, may form clusters that exhibit an excess of the second regionor second color relative to the first region or first color, asdiscussed above. The particles may be present, for example, in thebloodstream and/or within the skin of the subject.

In some cases, the particles, after delivery into the skin may give theappearance of a “tattoo” or a permanent mark within the skin, and thetattoo or other mark may be of any color and/or size. For instance, inone embodiment, anisotropic particles such as those described above thatare able to bind glucose may be delivered into the skin of a subject,and such particles, after deposition within the skin, may react to thepresence or absence of glucose by exhibiting a change in color. Theparticles may exhibit a color change based on the presence or absence ofglucose, and/or the concentration of glucose. For instance, theparticles may exhibit a first color (e.g., green) when not aggregated,and a second color (e.g., red or brown) when aggregated, or theparticles may be invisible when not aggregated, but visible (e.g.,exhibiting a color) when aggregated. The particles may be, for example,anisotropic particles having a first surface region having a first color(e.g., green) and a second surface region having a second color (e.g.,red), and the first surface region may contain a binding partner toglucose. At low levels of glucose, the particles may exhibit acombination of the first and second colors, while at higher levels ofglucose, the particles may exhibit more of the second color.

It should be noted that causing clustering of particles to occur is notlimited to only the exposure of particles to an analyte. In another setof embodiments, for example, the clustering or aggregation properties ofthe particles is externally controlled in some fashion. For instance, anelectrical, magnetic, and/or a mechanical force can be used to bring theparticles closer together and/or cause the particles to separate. Thus,in some cases, the application of an electrical, magnetic, and/or amechanical force to the particles causes the particles to exhibit achange in color. The clustering or aggregation of particles as discussedherein is not limited to generally spherical aggregations. In somecases, the particles may cluster onto a surface, or the particles may bealigned in some fashion relative to the surface due to an analyte orother external force.

In addition, it should be noted that the particles may contain reactionentities that are not necessarily binding partners to an analyte. Forinstance, there may be first particles containing a first reactionentity and a second reaction entity that reacts with the first reactionentity; when the particles are brought together in some fashion (e.g.,by exposure to an analyte or other chemical that is recognized bybinding partners on each of the particles, by the application of anelectrical, magnetic, and/or a mechanical force to bring the particlescloser together, etc.), the first and second reaction entities mayreact. As a specific example, the reaction between the first and secondreaction entities may be an endothermic or an exothermic reaction; thus,when the particles are brought together, a temperature change isproduced, which can be determined in some fashion. As another example, areaction between the first and second reactants may cause the release ofa material. In some cases, the material may be one that can be sensed bya subject, e.g., capsaicin, an acid, an allergen, or the like. Thus, thesubject may sense the change as a change in temperature, pain,itchiness, swelling, or the like.

In some cases, the particles may be suspended in a carrying fluid, e.g.,saline, or the particles may be contained within a matrix, e.g., aporous matrix that is or becomes accessible by interstitial fluid afterdelivery, or a hydrogel matrix, etc. For instance, the matrix may beformed from a biodegradable and/or biocompatible material such aspolylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), etc.,or other similar materials.

In some cases, the matrix may prevent or at least inhibit animmunological response by the subject to the presence of the particles,while allowing equilibration of analytes, etc. with the particles tooccur, e.g., if the matrix is porous. For instance, the pores of aporous matrix may be such that immune cells are unable to penetrate,while proteins, small molecules (e.g., glucose, ions, dissolved gases,etc.) can penetrate. The pores may be, for instance, less than about 5micrometers, less than about 4 micrometers, less than about 3micrometers, less than about 2 micrometers, less than about 1.5micrometers, less than about 1.0 micrometers, less than about 0.75micrometers, less than about 0.6 micrometers, less than about 0.5micrometers, less than about 0.4 micrometers, less than about 0.3micrometers, less than about 0.1 micrometers, less than about 0.07micrometers, and in other embodiments, or less than about 0.05micrometers. The matrix may comprise, for example, biocompatible and/orbiodegradable polymers such as polylactic and/or polyglycolic acids,polyanhydride, polycaprolactone, polyethylene oxide, polybutyleneterephthalate, starch, cellulose, chitosan, and/or combinations ofthese, and/or other materials such as agarose, collagen, fibrin, or thelike.

Thus, in one set of embodiments, particles are provided which can beanalogized to a light on an automotive dashboard, e.g., green fornormal, yellow for suspicious, slightly low or slightly high, and redfor abnormal. The subject then knows that they need to be seen, and thedegree of urgency, by appropriate medical personnel. The particles maybe placed and read at the site of detection. For example, the devicesmay provide a visual colorimetic signal, but other signals are possible,such as smell (released upon change in pH or temperature, for example),or tactile (shape change due to chemical reaction).

Signals from the particles can be used to generate a pattern or colorwhich is indicative of the presence and/or amount of analyte. Thedensity, shape, color, or intensity of the pattern or color may providea yes-no type answer or may be graduated to provide quantitativeamounts. This could also be effected by exposure to a pH or temperaturechange in some embodiments. Other patterns include, for example, + and −signs, arrows (e.g., up arrows or down arrows), faces (smiley, neutral,sad), etc., or the like.

In one set of embodiments, the skin surface may change in feel whenthere is a reaction. For example, shape memory polymers may say “OK”when the cholesterol level is below 150 mg/dl. These may change to ready“HIGH” when the cholesterol level exceeds 200 mg/dl. The device may beblank or lack definition at values between these levels.

The particles may change when reacted with analyte. This may result in asmell such as a food odor being released as a function of a pH ortemperature change. For instance, an encapsulated scent may be released.In some cases, FDA GRAS ingredients may be used as signals.

These may be applied to the skin to measure a change in temperatureindicative of disease or inflammation. In one embodiment, the device maybe colorless or a color indicative of normal temperature (for example,green), or the device will display a message such as “OK.” In the eventthe temperature exceeds a certain level, such as 101° F., the colorchanges (for example, yellow for caution or red for warning or critical)or the message changes (for example, if shape memory polymers are used)to read “HOT.” These are particularly useful in a setting such as a daycare, where there are a number of babies or young children to supervise,and fevers can occur rapidly.

In another embodiment, the particles may be used to measure a decreasein blood oxygen, or measure the amount of molecules such as glucose,cholesterol, triglycerides, cancer markers, or infectious agents, byproviding agents that specifically react with the molecules, and signalgenerating agents which produce signal in an amount correlated with theamounts of the molecules that react. As another non-limiting example,analogous to the temperature monitor, a pre-set level can be used tocreate a message that says “C high,” for example, or “insulin!” forexample, which effects a color change.

As discussed above, the devices may, instead of a color change ormessage change, change shape, emit a scent or flavor, or otherwisenotify the person of a need to seek further information. In some cases,this might be to seek medical attention where the indicator of adisorder can be confirmed and appropriate medical intervention obtained.In the case of temperature indicative of a fever, the caregiver mightmeasure the temperature using a standard thermometer. In the case of ahormone change, indicative of pregnancy or ovulation, an ELISA testmight be performed using a urine sample. In the case of high glucose,this could be confirmed using a standard glucose monitor and a bloodsample.

In another aspect, the particles may be delivered for cosmetic purposes,e.g., as a permanent or temporary tattoo. In some cases, the “tattoo” orparticles contained within the skin may be alterable by theadministration of an electrical, magnetic, and/or a mechanical force tothe subject. For instance, by applying such forces, the particles may becaused to cluster, which may result in a change in color, as discussedabove. Thus, one embodiment of the invention is directed to a cosmeticmark in the skin that can be altered by application of an externalstimulus, such as an electrical, magnetic, and/or a mechanical force,and/or a chemical applied to the skin (e.g., a chemical which is abinding partner of a species on the particle).

The tattoo (or other mark) present in the skin may have any function,e.g., as a decorative art, or as an identification system. For instance,a tattoo may be verified by applying a stimulus to the subject (e.g., anelectric field, a magnetic field, a mechanical force, a chemical, etc.),and confirming the tattoo by identifying a change in the mark, such as achange in color. The change in the mark may be permanent or temporary.As a specific example, a stimulus may be applied to anisotropicparticles containing a first region exhibiting a first color and asecond region exhibiting a second color. In the absence of the stimulus,the particles exhibit a blend of the first and second colors; however,under application of the stimulus, only one color may be exhibited asthe particles are aligned. This identification of a change in color maybe used, for example, artistically, or as an identifying mark. Asmentioned, in some cases, such a mark may be permanent or temporary. Asanother example, the particles may be invisible (e.g., non-aggregated)in the absence of a stimulus, but become visible (e.g., aggregated) whena stimulus is applied. In some cases, the particles change theirappearance while the stimulus is applied, but revert to their originalappearance once the stimulus is removed; in other cases, however, theparticles may be able to retain their altered appearance for some timefollowing removal of the stimulus, and in some cases, the particlespermanently retain their altered appearance.

In another aspect, the present invention is directed to a kit includingone or more of the compositions previously discussed, e.g., a kitincluding a particle, a kit including a device for the delivery and/orwithdrawal of fluid from the skin, a kit including a device able tocreate a pooled region of fluid within the skin of a subject, a kitincluding a device able to determine a fluid, or the like. An example ofa kit containing more than one device of the invention is illustrated inFIG. 11B, with kit 150 containing devices 152. A “kit,” as used herein,typically defines a package or an assembly including one or more of thecompositions or devices of the invention, and/or other compositions ordevices associated with the invention, for example, as previouslydescribed. For example, in one set of embodiments, the kit may include adevice and one or more compositions for use with the device. Each of thecompositions of the kit, if present, may be provided in liquid form(e.g., in solution), or in solid form (e.g., a dried powder). In certaincases, some of the compositions may be constitutable or otherwiseprocessable (e.g., to an active form), for example, by the addition of asuitable solvent or other species, which may or may not be provided withthe kit. Examples of other compositions or components associated withthe invention include, but are not limited to, solvents, surfactants,diluents, salts, buffers, emulsifiers, chelating agents, fillers,antioxidants, binding agents, bulking agents, preservatives, dryingagents, antimicrobials, needles, syringes, packaging materials, tubes,bottles, flasks, beakers, dishes, frits, filters, rings, clamps, wraps,patches, containers, tapes, adhesives, and the like, for example, forusing, administering, modifying, assembling, storing, packaging,preparing, mixing, diluting, and/or preserving the compositionscomponents for a particular use, for example, to a sample and/or asubject.

A kit of the invention may, in some cases, include instructions in anyform that are provided in connection with the compositions of theinvention in such a manner that one of ordinary skill in the art wouldrecognize that the instructions are to be associated with thecompositions of the invention. For instance, the instructions mayinclude instructions for the use, modification, mixing, diluting,preserving, administering, assembly, storage, packaging, and/orpreparation of the compositions and/or other compositions associatedwith the kit. In some cases, the instructions may also includeinstructions for the delivery and/or administration of the compositions,for example, for a particular use, e.g., to a sample and/or a subject.The instructions may be provided in any form recognizable by one ofordinary skill in the art as a suitable vehicle for containing suchinstructions, for example, written or published, verbal, audible (e.g.,telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) orelectronic communications (including Internet or web-basedcommunications), provided in any manner.

In some embodiments, the present invention is directed to methods ofpromoting one or more embodiments of the invention as discussed herein.As used herein, “promoted” includes all methods of doing businessincluding, but not limited to, methods of selling, advertising,assigning, licensing, contracting, instructing, educating, researching,importing, exporting, negotiating, financing, loaning, trading, vending,reselling, distributing, repairing, replacing, insuring, suing,patenting, or the like that are associated with the systems, devices,apparatuses, articles, methods, compositions, kits, etc. of theinvention as discussed herein. Methods of promotion can be performed byany party including, but not limited to, personal parties, businesses(public or private), partnerships, corporations, trusts, contractual orsub-contractual agencies, educational institutions such as colleges anduniversities, research institutions, hospitals or other clinicalinstitutions, governmental agencies, etc. Promotional activities mayinclude communications of any form (e.g., written, oral, and/orelectronic communications, such as, but not limited to, e-mail,telephonic, Internet, Web-based, etc.) that are clearly associated withthe invention.

In one set of embodiments, the method of promotion may involve one ormore instructions. As used herein, “instructions” can define a componentof instructional utility (e.g., directions, guides, warnings, labels,notes, FAQs or “frequently asked questions,” etc.), and typicallyinvolve written instructions on or associated with the invention and/orwith the packaging of the invention. Instructions can also includeinstructional communications in any form (e.g., oral, electronic,audible, digital, optical, visual, etc.), provided in any manner suchthat a user will clearly recognize that the instructions are to beassociated with the invention, e.g., as discussed herein.

U.S. Provisional Patent Application Ser. No. 61/058,796, filed Jun. 4,2008, entitled “Compositions and Methods for Diagnostics, Therapies, andOther Applications,” by D. Levinson, is incorporated herein byreference. Also incorporated herein by reference are U.S. ProvisionalPatent Application Ser. No. 61/163,733, filed on Mar. 26, 2009, entitled“Determination of Tracers within Subjects,” by D. Levinson; U.S.Provisional Patent Application Ser. No. 61/163,750, filed on Mar. 26,2009, entitled “Monitoring of Implants and Other Devices,” by D.Levinson, et al.; U.S. Provisional Patent Application Ser. No.61/058,682, filed on Mar. 26, 2009, entitled “Compositions and Methodsfor Diagnostics, Therapies, and other Applications,” by D. Levinson;U.S. Provisional Patent Application Ser. No. 61/163,793, filed Mar. 26,2009, entitled “Compositions and Methods for Diagnostics, Therapies, andOther Applications,” by D. Levinson; U.S. patent application Ser. No.12/478,756, filed Jun. 4, 2009, entitled “Compositions and Methods forDiagnostics, Therapies, and Other Applications”; International PatentApplication No. PCT/US09/046333, filed Jun. 4, 2009, entitled“Compositions and Methods for Diagnostics, Therapies, and OtherApplications”; U.S. Provisional Patent Application Ser. No. 61/163,710,filed Mar. 26, 2009, entitled “Systems and Methods for Creating andUsing Suction Blisters or Other Pooled Regions of Fluid within theSkin”; U.S. Provisional Patent Application Ser. No. 61/156,632, filedMar. 2, 2009, entitled “Oxygen Sensor”; U.S. Provisional PatentApplication Ser. No. 61/269,436, filed Jun. 24, 2009, entitled “Devicesand Techniques associated with Diagnostics, Therapies, and OtherApplications, Including Skin-Associated Applications”; U.S. ProvisionalPatent Application Ser. No. 61/163,791, filed on Mar. 26, 2009, entitled“Compositions and Methods for Rapid One-Step Diagnosis,” by D. Levinson;U.S. Provisional Patent Application Ser. No. 61/257,731, filed Nov. 3,2009, entitled “Devices and Techniques Associated with Diagnostics,Therapies, and Other Applications, Including Skin-AssociatedApplications”; and U.S. Provisional Patent Application Ser. No.61/294,543, filed Jan. 13, 2010, entitled “Blood Sampling Device andMethod.” Also incorporated herein by reference are the following U.S.patent applications being filed on even date herewith: “Oxygen Sensor,”by Levinson, et al.; “Systems and Methods for Creating and Using SuctionBlisters or Other Pooled Regions of Fluid within the Skin,” by Levinson,et al.; and “Techniques and Devices Associated with Blood Sampling,” byLevinson et al.

In one aspect of the present invention, methods of forming particlessuch as those described herein are provided. For instance, in one set ofembodiments, electrospraying or electrospinning techniques are used toprepare particles. In some cases, two or more fluid streams (includingliquid jets) are combined together such that the two or more fluidstreams contact over spatial dimensions sufficient to form a compositestream. In some cases, there is little or no mixing of the two or morefluid streams within the composite stream. In some variations, the fluidstreams are electrically conductive, and in certain cases, a cone-jetmay be formed by combining the two or more fluid streams under theinfluence of an electric field.

In some cases, the composite stream is directed at a substrate, e.g., bythe application of a force field such as an electric field. Forinstance, if the composite stream is charged, an electric field may beused to urge the composite stream towards a substrate. The compositestream may be continuous or discontinuous in some cases, e.g., forming aseries of droplets (which may be spherical or non-spherical). In somecases, the composite stream is hardened prior to and/or upon contactwith the substrate. For example, the composite stream may be urgedtowards the substrate under conditions in which at least a portion ofthe composite stream (e.g., a solvent) is able to evaporate, causing theremaining stream to harden and/or precipitate, e.g., to form particles,spheres, rods, fibers, or the like. In some variations, the compositestream fragments in droplets that can lead to particle, sphere, rod,and/or fiber formation.

Additional examples of techniques for forming such particles or fiberscan be found in U.S. patent application Ser. No. 11/272,194, filed Nov.10, 2005, entitled “Multi-Phasic Nanoparticles,” by Lahann, et al.,published as U.S. Patent Application Publication No. 2006/0201390 onSep. 14, 2006; or priority to U.S. patent application Ser. No.11/763,842, filed Jun. 15, 2007, entitled “Multiphasic BiofunctionalNano-Components and Methods for Use Thereof,” by Lahann, published asU.S. Patent Application Publication No. 2007/0237800 on Oct. 11, 2007,each of which is incorporated herein by reference.

In one set of embodiments, solvent evaporation techniques may be used.In one embodiment, a polymer may be dissolved in a volatile organicsolvent, such as methylene chloride. Drugs or other suitable species areadded to the solution, and the mixture is suspended in an aqueoussolution that contains a surface active agent such as poly(vinylalcohol). The resulting emulsion can be stirred until most of theorganic solvent evaporated, leaving solid particles. The resultingparticles may be washed with water and dried overnight in a lyophilizer.Particles with different sizes or morphologies can be obtained by thismethod. This method is useful for relatively stable polymers likepolyesters and polystyrene.

In another set of embodiments, solvent removal techniques may be used,e.g., for polymers such as polyanhydrides. In one embodiment, a polymermay be dissolved in a volatile organic solvent like methylene chloride.The mixture can be suspended by stirring in an organic oil (such assilicon oil) to form an emulsion. This can be used to make particlesfrom polymers with high melting points and different molecular weights.Particles that range, for example, between 1-2000 microns, 1-1000microns, 1-500 microns, 1-300 microns, 1-100 microns, 1-30 microns, 1-10microns, etc. in diameter can be obtained by this procedure. Theexternal morphology of spheres produced with this technique may becontrolled by controlling the type of polymer used.

In yet another set of embodiments, spray-drying techniques may be used.In one embodiment, a polymer is dissolved in organic solvent. Thesolution or the dispersion is then spray-dried. Particles rangingbetween, for example, between 1-2000 microns, 1-1000 microns, 1-500microns, 1-300 microns, 1-100 microns, 1-30 microns, 1-10 microns, etc.in diameter can be obtained with a morphology which depends on the typeof polymer used.

In still another set of embodiments, interfacial polycondensationtechniques may be used. In one embodiment, a monomer is dissolved in asolvent. A second monomer is dissolved in a second solvent (typicallyaqueous) which is immiscible with the first. An emulsion may be formedby suspending the first solution through stirring in the secondsolution. Once the emulsion is stabilized, an initiator can be added tothe aqueous phase causing interfacial polymerization at the interface ofeach droplet of emulsion.

In yet another set of embodiments, phase inversion techniques may beused. In one set of embodiments, particles can be formed from polymersusing a phase inversion method wherein a polymer is dissolved in asolvent and the mixture is poured into a non-solvent for the polymer, tospontaneously produce particles under favorable conditions. The methodcan be used to produce particles in a wide range of diameters,including, for example, about 100 nanometers to about 10 microns.Examples of polymers which can be used include polyvinylphenol andpolylactic acid. In some cases, the polymer can be dissolved in anorganic solvent and then contacted with a non-solvent, which causesphase inversion of the dissolved polymer to form particles, optionallyincorporating an antigen or other substance.

In still another set of embodiments, phase separation techniques may beused. In one set of embodiments, the polymer is dissolved in a solventto form a polymer solution. While continually stirring, a nonsolvent forthe polymer may be added to the solution to decrease the polymer'ssolubility. Depending on the solubility of the polymer in the solventand nonsolvent, the polymer may precipitate and/or phase separate into apolymer-rich and a polymer-poor phase. Under proper conditions, thepolymer in the polymer-rich phase may migrate to the interface with thecontinuous phase, forming particles.

In yet another set of embodiments, spontaneous emulsification techniquescan be used. One set of embodiments involves solidifying emulsifiedliquid polymer droplets by changing temperature, evaporating solvent,and/or adding chemical cross-linking agents. In still another set ofembodiments, hot melt techniques may be used.

In some cases, the particles may comprise a gel. For instance, in oneset of embodiments, particles made of gel-type polymers, such asalginate and hyaluronic acid, can be produced through ionic gelationtechniques. In one embodiment, polymers can be first dissolved in anaqueous solution and then extruded through a droplet forming device,which in some instances employs a flow of nitrogen and/or other gases tobreak off the droplet. A slowly stirred (approximately 100-170 RPM)ionic hardening bath may be positioned below the extruding device tocatch the forming droplets. The particles are left to incubate in thebath to allow gelation to occur. Particle size may be controlled, forexample, by using various size extruders or varying nitrogen gas orpolymer solution flow rates. In one embodiment, chitosan particles canbe prepared by dissolving the polymer in acidic solution andcrosslinking it with tripolyphosphate. In another embodiment,carboxymethyl cellulose (CMC) nanoparticles can be prepared bydissolving the polymer in acid solution and precipitating thenanoparticle with lead ions. In some cases where negatively chargedpolymers (e.g., alginate, CMC) are used, positively charged ligands(e.g., polylysine, polyethyleneimine) of different molecular weights canbe ionically attached.

Other methods known in the art that can be used to prepare nanoparticlesinclude, but are not limited to, polyelectrolyte condensation, singleand double emulsion (probe sonication), nanoparticle molding, orelectrostatic self-assembly (e.g., polyethylene imine-DNA or liposomes).

In some cases, the particles may include functional groups used to bindor complex the analyte, and such functional groups can be introducedprior to particle formation (e.g., monomers can be functionalized withone or more functional groups for binding or complexing the analyte) orthe functional groups can be introduced after particle formation (e.g.,by functionalizing the surface of the microparticle with reactivefunctional groups). The particles may optionally have encapsulatedtherein one or more core materials. In one embodiment, the particles maybe present in an effective amount to provide a signal detectable to theuser without the need for additional equipment. For example, thearticles should be present in an effective amount to provide a change intaste, smell, shape, and/or color upon binding or complexing the analytethat is easily detectable by the user.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is: 1-47. (canceled)
 48. A device for withdrawing bloodfrom the skin and/or from beneath the skin of a subject, the devicecomprising: a vacuum chamber having an internal pressure less thanatmospheric pressure before blood is withdrawn into the device andbefore the device is applied to the skin of the subject; a storagechamber, separate from the vacuum chamber and the recess, for receivingblood withdrawn into the recess when a negative pressure is applied tothe skin of the subject; and a sensor for determining a condition of theblood withdrawn into the device.
 49. The device of claim 48, furthercomprising a microfluidic channel, discrete from the vacuum chamber andthe storage chamber and having a smaller cross-sectional area than thecross-sectional area of the storage chamber, wherein the vacuum chamberis in fluid communication with the storage chamber via the microfluidicchannel.
 50. The device of claim 48, further comprising a plurality ofmicroneedles for accessing the blood from and/or through the skin of thesubject.
 51. The device of claim 50, further comprising a supportstructure constructed and arranged to move the plurality of microneedlesinto contact with the skin, and to withdrawal the plurality ofmicroneedles from the skin after contact with the skin.
 52. The deviceof claim 48, further comprising a reaction entity contained within thestorage chamber able to react with an analyte contained within theblood, wherein a product of the reaction entity reacted with the analyteis determinable by the sensor.
 53. The device of claim 48, wherein thevacuum chamber does not have a variable volume.
 54. The device of claim48, further comprising a signal generator for generating a signalrelating to the blood determined by the sensor.
 55. The device of claim48, further comprising a display able to display a result of thedetermination of the analyte by the sensor.
 56. A device for withdrawingblood from the skin and/or from beneath the skin of a subject, thedevice comprising: a support structure constructed and arranged to bepositioned proximate the skin of the subject; a plurality ofmicroneedles, associated with the support structure, for accessing bloodfrom and/or through the skin of the subject; and a sensor fordetermining a condition of the blood withdrawn into the device, whereinthe support structure is constructed and arranged to move the pluralityof microneedles into contact with the skin, and to withdrawal theplurality of microneedles from the skin after contact with the skin. 57.The device of claim 56, wherein the support structure is able to apply avacuum to the skin.
 58. The device of claim 57, wherein the supportstructure comprises a vacuum chamber having an internal pressure lessthan atmospheric pressure before blood is withdrawn into the device andbefore the device is applied to the skin of the subject.
 59. The deviceof claim 58, further comprising a storage chamber, separate from thevacuum chamber.
 60. The device of claim 59, further comprising amicrofluidic channel, discrete from the vacuum chamber and the storagechamber and having a smaller cross-sectional area than thecross-sectional area of the storage chamber, wherein the vacuum chamberis in fluid communication with the storage chamber via the microfluidicchannel.
 61. The device of claim 56, wherein the sensor is positioned tosense at least a portion of the storage chamber.
 62. The device of claim56, wherein the plurality of microneedles is able to move relative tothe support structure.
 63. The device of claim 56, wherein the pluralityof microneedles comprises solid microneedles.
 64. The device of claim56, further comprising a signal generator for generating a signalrelating to the blood determined by the sensor.
 65. The device of claim56, further comprising a display able to display a result of thedetermination of the analyte by the sensor.
 66. The device of claim 56,wherein the support structure is constructed and arranged to move theplurality of microneedles into contact with the skin, and to withdrawalthe plurality of microneedles from the skin after contact with the skin.67. The device of claim 56, further comprising a reaction entity able toreact with an analyte contained within the blood, wherein a product ofthe reaction entity reacted with the analyte is determinable by thesensor.